Process for immobilizing enzymes to the cell wall of a microbial cell by producing a fusion protein

ABSTRACT

A method is provided for immobilizing an enzyme, comprising immobilizing the enzyme or a functional part thereof to the cell wall of a microbial cell using recombinant DNA techniques. The enzyme is immobilized by linking it to the C-terminal part of a protein that ensures anchoring in the cell wall. Also provided is a recombinant polynucleotide comprising a structural gene encoding an enzyme protein, a part of a gene encoding the C-terminal part of a protein capable of anchoring in a eukaryotic or prokaryotic cell wall, as well as a signal sequence, in addition to a chimeric protein encoded by the recombinant polynucleotide and a vector and a microorganism containing the polynucleotide. The microorganism is suitable for carrying out enzymatic processes on an industrial scale.

The present invention is in the field of conversion processes usingimmobilized enzymes, produced by genetic engineering.

BACKGROUND OF THE INVENTION

In the detergent, personal care and food products industry there is astrong trend towards natural ingredients of these products and toenvironmentally acceptable production processes. Enzymic conversions arevery important for fulfilling these consumer demands, as these processescan be completely natural. Moreover enzymic processes are very specificand consequently will produce minimum amounts of waste products. Suchprocesses can be carried out in water at mild temperatures andatmospheric pressure. However enzymic processes based on free enzymesare either quite expensive due to the loss of enzymes or requireexpensive equipment, like ultra-membrane systems to entrap the enzyme.

Alternatively enzymes can be immobilized either physically orchemically. The latter method has often the disadvantage that couplingis carried out using non-natural chemicals and in processes that are notattractive from an environmental point of view. Moreover chemicalmodification of enzymes is nearly always not very specific, which meansthat coupling can affect the activity of the enzyme negatively. Physicalimmobilization can comply with consumer demands, however also physicalimmobilization may affect the activity of the enzyme in a negative way.Moreover, a physically immobilized enzyme is in equilibrium with freeenzyme, which means that in continuous reactors, according to the lawsof thermodynamics, substantial losses of enzyme are unavoidable.

There are a few publications on immobilization of enzymes to microbialcells (see reference 1). The present invention provides a method forimmobilizing enzymes to cell walls of microbial cells in a very preciseway. Additionally, the immobilization does not require any chemical orphysical coupling step and is very efficient. Some extracellularproteins are known to have special functions which they can perform onlyif they remain bound to the cell wall of the host cell. Often this typeof protein has a long C-terminal part that anchors it in the cell wall.These C-terminal parts have very special amino acid sequences. A typicalexample is anchoring via C-terminal sequences enriched in proline (seereference 2). Another mechanism to anchor proteins in cell walls is thatthe protein has a glycosyl-phosphatidyl-inositol (GPI) anchor (seereference 3) and that the C-terminal part of the protein contains asubstantial number of potential serine and threonine glycosylationsites. O-Glycosylation of these sites gives a rod-like conformation tothe C-terminal part of these proteins. Another feature of thesemanno-proteins is that they seem to be linked to the glucan in the cellwall of lower eukaryotes, as they cannot be extracted from the cell wallwith SDS, but can be liberated by glucanase treatment.

SUMMARY OF THE INVENTION

The present invention provides a method for immobilizing an enzyme,which comprises the use of recombinant DNA techniques for producing anenzyme or a functional part thereof linked to the cell wall of a hostcell, preferably a microbial cell, and whereby the enzyme or functionalfragment thereof is localized at the exterior of the cell wall.Preferably the enzyme or the functional part thereof is immobilized bylinking to the C-terminal part of a protein that ensures anchoring inthe cell wall.

In one embodiment of the invention a recombinant polynucleotide isprovided comprising a structural gene encoding a protein providingcatalytic activity and at least a part of a gene encoding a proteincapable of anchoring in a eukaryotic or prokaryotic cell wall, said partencoding at least the C-terminal part of said anchoring protein.Preferably the polynucleotide further comprises a sequence encoding asignal peptide ensuring secretion of the expression product of thepolynucleotide. Such signal peptide can be derived from aglycosyl-phosphatidyl-inositol (GPI) anchoring protein, α-factor,α-agglutinin, invertase or inulinase, α-amylase of Bacillus, or aproteinase of lactic acid bacteria. The DNA sequence encoding a proteincapable of anchoring in the cell wall can encode α-agglutinin, AGA1(a-agglutinin) FLO1 (flocculation protein) or the Major Cell WallProtein of lower eukaryotes, or a proteinase of lactic acid bacteria.The recombinant polynucleotide is operably linked to a promoter,preferably an inducible promoter. The DNA sequence encoding a proteinproviding catalytic activity can encode a hydrolytic enzyme, e.g. alipase, or an oxidoreductase, e.g. an oxidase. Another embodiment of theinvention relates to a recombinant vector comprising a polynucleotide asdescribed above. If such vector contains a DNA sequence encoding aprotein providing catalytic activity, which protein exhibits saidcatalytic activity when present in a multimeric form, said vector canfurther comprise a second polynucleotide comprising a structural geneencoding the same protein providing catalytic activity combined with asequence encoding a signal peptide ensuring secretion of the expressionproduct of said second polynucleotide, said second polynucleotide beingoperably linked to a regulatable promoter, preferably an inducible orrepressible promoter.

A further embodiment of the invention relates to a chimeric proteinencoded by a polynucleotide as described above.

Still another embodiment is a host cell, preferably a microorganism,containing a polynucleotide as described above or a vector as describedabove. If the protein providing catalytic activity exhibits saidcatalytic activity when present in a multimeric form, said host cell ormicroorganism can further comprise a second polynucleotide comprising astructural gene encoding the same protein providing catalytic activitycombined with a sequence encoding a signal peptide ensuring secretion ofthe expression product of said second polynucleotide, said secondpolynucleotide being operably linked to a regulatable promoter,preferably an inducible or repressible promoter, and said secondpolynucleotide being present either in another vector or in thechromosome of said microorganism. Preferably the host cell ormicroorganism has at least one of said polynucleotides integrated in itschromosome. As a result of culturing such host cell or microorganism theinvention provides a host cell, preferably a microorganism, having aprotein as described above immobilized on its cell wall. The host cellor microorganism can be a lower eukaryote, in particular a yeast.

The invention also provides a process for carrying out an enzymaticprocess by using an immobilized catalytically active protein, wherein asubstrate for said catalytically active protein is contacted with a hostcell or microorganism according to the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: DNA sequence of the 6057 bp HindIII fragment containing thecomplete AGα1 gene of S. cerevisiae (see SEQ ID NO: 1). The position ofthe unique NheI site and the HindIII site used for the describedconstructions is specified in the header.

FIG. 2: Schematic presentation of the construction of pUR2969. Therestriction sites for endonucleases used are shown. Abbreviations used:AG-alpha-1: Gene expressing α-agglutinin from S. cerevisiae

amp: β-lactamase resistance gene

PGKp: phosphoglyceratekinase promoter

PGKt: terminator of the same gene.

FIG. 3: α-Galactosidase activity of S. cerevisiae MT302/1C cells andculture fluid transformed with pSY13 during batch culture:

A: U/l α-galactosidase per time; the OD₅₃₀ is also shown

B: α-galactosidase activity of free and bond enzyme expressed inU/OD₅₃₀.

FIG. 4: α-Galactosidase activity of S. cerevisiae MT302/1C cells andculture fluid transformed with pUR2969 during batch culture:

A: U/l α-galactosidase per time; the OD₅₃₀ is also shown

B: α-galactosidase activity of free and bond enzyme expressed inU/OD₅₃₀.

FIG. 5: Western analysis with anti α-galactosidase serum ofextracellular fractions of cells of exponential phase (OD₅₃₀ =2). Theanalyzed fractions are equivalent to 4 mg cell walls, (fresh weight):

A: MT302/1C expressing α-galactosidase,

lane 1, growth medium

lane 2, SDS extract of isolated cell walls

lane 3, glucanase extract of SDS extracted cell walls;

B: MT302/1C expressing α-Gal-AGα1 fusion protein,

lane 1, growth medium

lane 2, SDS extract of isolated cell walls

lane 3, glucanase extract of SDS-extracted cell walls

lane 4: Endo-H treated glucanase extract.

FIG. 6: Immunofluorescent labelling (anti α-galactosidase) of MT302/1Ccells in the exponential phase (OD₅₃₀ =2) expressing theα-Gal-α-agglutinin fusion protein.

Phase micrograph of intact cells A: overview B: detail.

FIG. 7: Schematic presentation of the construction of pUR2970A,pUR2971A, pUR2972A, and pUR2973. The restriction sites for endonucleasesused are indicated in the figure. PCR oligonucleotide sequences arementioned in the text.

Abbreviations used: AGa1 cds: coding sequence of α-agglutinin

a-AGG=AGa1: Gene expressing α-agglutinin from S. cerevisiae

amp: β-lactamase resistance gene

lipolase: lipase gene of Humicola

a-MF: prepro-α-mating factor sequence

Pgal7=GAL7: GAL7 promoter

invSS: SUC2 signal sequence

a-gal: α-galactosidase gene

LEU2d: truncated promoter of LEU2 gene;

LEU2: LEU2 gene with complete promoter sequence.

FIG. 8: DNA sequence of a fragment containing the complete codingsequence of lipase B of Geotrichum candidum strain 335426 (see SEQ IDNO: 11). The sequence of the mature lipase B starts at nucleotide 97 ofthe given sequence. The coding sequence starts at nucleotide 40 (ATG).

FIG. 9: Schematic presentation of the construction of pUR2975 andpUR2976. The restriction sites for endonucleases used are shown.Abbreviations used:

a-AGG: Gene expressing α-agglutinin from S. cerevisiae

amp: β-lactamase resistance gene

invSS: SUC2 signal sequence

LEU2d: truncated promoter LEU2 gene

Pgal7=GAL7: GAL7 promoter

a-MF: prepro-α-mating factor sequence

lipolase: lipase gene of Humicola

lipaseB: lipaseB gene of Geotrichum candidum.

FIG. 10: Schematic presentation of the construction of pUR2981 andpUR2982. The restriction sites for endonucleases used are shown.Abbreviations used:

a-AGG=AG-alpha 1: Gene expressing α-agglutinin from S. cerevisiae

mucor lipase: lipase gene of Rhizomucor miehei

Pgal7=GAL7: GAL7 promoter

a-MF: prepro-α-mating factor sequence

amp: β-lactamase resistance gene;

2u: 2 μm sequence

invSS: SUC2 signal sequence

lipolase: lipase gene of Humicola

LEU2d: truncated promoter LEU2 gene

LEU2: LEU2 gene with complete promoter sequence.

FIG. 11: DNA sequence (2685 bases) of the 894 amino acids coding part ofthe FLO1 gene (see SEQ ID NO: 21), the given sequence starts with thecodon for the first amino acid and ends with the stop codon.

FIG. 12: Schematic presentation of plasmid pUR2990. Some restrictionsites for endonucleases relevant for the given cloning procedure areshown.

FIG. 13: Schematic presentation of plasmid pUR7034.

FIG. 14: Schematic presentation of plasmid pUR2972B.

FIG. 15: Immunofluorescent labelling (anti-lipolase) of SU10 cells inthe exponential phase (OD₅₃₀ =0.5) expressing the lipolase/-α-agglutininfusion protein.

A: phase micrograph B: matching fluorescent micrograph

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for immobilizing an enzyme,comprising immobilizing the enzyme or a functional part thereof to thecell wall of a host cell, preferably a microbial cell, using recombinantDNA techniques. In particular, the C-terminal part of a protein thatensures anchoring in the cell wall is linked to an enzyme or thefunctional part of an enzyme, in such a way that the enzyme is localizedon or just above the cell surface. In this way immobilized enzymes areobtained on the surface of cells. The linkage is performed at gene leveland is characterized in that the structural gene coding for the enzymeis coupled to at least part of a gene encoding an anchor-protein in sucha way that in the expression product the enzyme is coupled at itsC-terminal end to the C-terminal part of an anchor-protein. The chimericenzyme is preferably preceded by a signal sequence that ensuresefficient secretion of the chimeric protein.

Thus the invention relates to a recombinant polynucleotide comprising astructural gene encoding a protein providing catalytic activity and atleast a part of a gene encoding a protein capable of anchoring in aeukaryotic or prokaryotic cell wall, said part encoding at least theC-terminal part of said anchoring protein. The length of the C-terminalpart of the anchoring protein may vary. Although the entire structuralprotein could be used, it is preferred that only a part is used, leadingto a more efficient exposure of the enzyme protein in the mediumsurrounding the cell. The anchoring part of the anchoring protein shouldpreferably be entirely present. As an example, about the C-terminal halfof the anchoring protein could be used. Preferably, the polynucleotidefurther comprises a sequence encoding a signal peptide ensuringsecretion of the expression product of the polynucleotide. The signalpeptide can be derived from a GPI anchoring protein, α-factor,α-agglutinin, invertase or inulinase, α-amylase of Bacillus, or aproteinase of lactic acid bacteria. The protein capable of anchoring inthe cell wall is preferably selected form the group of α-agglutinin,AGA1, FLO1 (flocculation protein) or the Major Cell Wall Protein oflower eukaryotes, or a proteinase of lactic acid bacteria. Thepolynucleotide of the invention is preferably operably linked to apromoter, preferably a regulatable promoter, especially an induciblepromoter.

The invention also relates to a recombinant vector containing thepolynucleotide as described above, and to a host cell containing thispolynucleotide, or this vector. In a particular case, wherein theprotein providing catalytic activity exhibits said catalytic activitywhen present in a multimeric form, such as may be the case withoxidoreductases, dimerisation or multimerisation of the monomers mightbe a prerequisite for activity. The vector and/or the host cell can thenfurther comprise a second polynucleotide comprising a structural geneencoding the same protein providing catalytic activity combined with asequence encoding a signal peptide ensuring secretion of the expressionproduct of said second polynucleotide, said second polynucleotide beingoperably linked to a regulatable promoter, preferably an inducible orrepressible promoter. Expression and secretion of the secondpolynucleotide after expression and secretion of the firstpolynucleotide will then result in the formation of an active multimeron the exterior of the cell wall. The host cell or microorganismpreferably contains the polynucleotide described above, or at least oneof said polynucleotides in the case of a combination, integrated in itschromosome.

The present invention relates in particular to lower eukaryotes likeyeasts that have very stable cell walls and have proteins that are knownto be anchored in the cell wall, e.g. α-agglutinin or the product ofgene FLO1. Suitable yeasts belong to the genera Candida, Debaryomyces,Hansenula, Kluyveromyces, Pichia and Saccharomyces. Also fungi,especially Aspergillus, Penicillium and Rhizopus can be used. Forcertain applications also prokaryotes are applicable.

For yeasts the present invention deals in particular with genes encodingchimeric enzymes consisting of:

a. the signal sequence e.g. derived from the α-factor-, the invertase-,the α-agglutinin- or the inulinase genes;

b. structural genes encoding hydrolytic enzymes such as α-galactosidase,proteases, peptidases, pectinases, pectylesterase, rhamnogalacturonase,esterases and lipases, or non-hydrolytic enzymes such as oxidases; and

c. the C-terminus of typically cell wall bound proteins such asα-agglutinin (see reference 4), AGA1 (see reference 5) and FLO1 (see thenon-prior published reference 6).

The expression of these genes can be under the control of a constitutivepromoter, but more preferred are regulatable, i.e. repressible orinducible promoters such as the GAL7 promoter for Saccharomyces, or theinulinase promoter for Kluyveromyces or the methanol-oxidase promoterfor Hansenula.

Preferably the constructs are made in such a way that the new geneticinformation is integrated in a stable way in the chromosome of the hostcell.

The invention further relates to a host cell, in particular amicroorganism, having the chimeric protein described above immobilizedon its cell wall. It further concerns the use of such microorganisms forcarrying out an enzymatic process by contacting a substrate for theenzyme with the microorganism. Such a process may be carried out e.g. ina packed column, wherein the microorganisms may be supported on solidparticles, or in a stirred reactor. The reaction may be aqueous ornon-aqueous. Where necessary, additives necessary for the performance ofthe enzyme, e.g. a co-factor, may be introduced in the reaction medium.

After repeated usage of the naturally immobilized enzyme system inprocesses, the performance of the system may decrease. This is causedeither by physical denaturation or by chemical poisoning or detachmentof the enzyme. A particular feature of the present invention is thatafter usage the system can be recovered from the reaction medium bysimple centrifugation or membrane filtration techniques and that thethus collected cells can be transferred to a recovery medium in whichthe cells revive quickly and concomitantly produce the chimeric protein,thus ensuring that the surface of the cells will be covered by fullyactive immobilized enzyme. This regeneration process is simple and cheapand therefore will improve the economics of enzymic processes and mayresult in a much wider application of processes based on immobilizedenzyme systems.

However, by no means the present invention is restricted to thereusability of the immobilized enzymes.

The invention will be illustrated by the following examples without thescope of the invention being limited thereto.

EXAMPLE 1 Immobilized α-Galactosidase/α-Agglutinin on the Surface of S.cerevisiase.

The gene encoding α-agglutinin has been described by Lipke et al. (seereference 4). The sequence of a 6057 bp HindIII insert in pTZ18R,containing the whole AGα1 gene is given in FIG. 1. The coding sequenceexpands over 650 amino acids, including a putative signal sequencestarting at nucleotide 3653 with ATG. The unique NheI site cuts the DNAat position 988 of the given coding sequence within the coding part ofamino acid 330, thereby separating the α-agglutinin into an N-terminaland a C-terminal part of about same size.

Through digestion of pUR2968 (see FIG. 2) with NheI/HindIII a 1.4 kbfragment was released, containing the sequence information of theputative cell wall anchor. For the fusion to α-galactosidase the plasmidpSY16 was used, an episomal vector based on YEplac 181, harboring theα-galactosidase sequence preceded by the SUC2 invertase signal sequenceand placed between the constitutive PGK promoter and PGK terminator. TheStyI site, present in the last nine base-pairs of the open reading frameof the α-galactosidase gene, was ligated to the NheI site of the AGα1gene fragment. To ensure the in frame fusion, the StyI site was filledin and the 5' overhang of the NheI site was removed, prior to ligationinto the StyI/HindIII digested pSY13 (see FIG. 2).

To verify the correct assembly of the new plasmid, the shuttle vectorwas transformed into E. coli JM109 (recA1 supE44 endA1 hsdR17 gyrA96relA1 thi ▴(lac-proAB) F' [traD36 proAB⁺ lacl^(q) lacZ▴M15]) (seereference 7) by the transformation protocol described by Chung et al.(see reference 8). One of the positive clones, designated pUR2969, wasfurther characterized, the DNA isolated and purified according to theQuiagen protocol and subsequently characterized by DNA sequencing. DNAsequencing was mainly performed as described by Sanger et al. (seereference 9), and Hsiao (see reference 10), here with the Sequenaseversion 2.0 kit from United States Biochemical Company, according to theprotocol with T7 DNA polymerase (Amersham International plc) and [³⁵S]dATPαS (Amersham International plc: 370 MBq/ml; 22 TBq/mmol).

This plasmid was then transformed into S. cerevisiae strain MT302/1Caccording to the protocol from Klebe et al. (see reference 11).

Yeast transformants were selected on selective plates, lacking leucine,on with 40 μl (20 mg/ml DMF). X-α-Gal(5-bromo-4-chloro-3-indolyl-α-D-glucose, Boehringer Mannheim) wasspread, to directly test for α-galactosidase activity (see reference12). To demonstrate the expression, secretion, localization and activityof the chimeric protein the following analyses were performed:

1. Expression and Secretion

S. cerevisiae strain MT302/1C was transformed with either plasmid pSY13containing the α-galactosidase gene of Cyamopsis tetragonoloba orplasmid pUR2969 containing the α-galactosidase/α-agglutinin fusionconstruct. During batch culture α-galactosidase activities weredetermined for washed cells and growth medium. The results are given inFIG. 3 and FIG. 4. The α-galactosidase expressed from yeast cellscontaining plasmid pSY13 was almost exclusively present in the growthmedium (FIG. 3A), whereas the α-galactosidase-α-agglutinin fusionprotein was almost exclusively cell associated (FIG. 4A). Moreover, theimmobilized, cell wall-associated, α-galactosidase-α-agglutinin fusionenzyme had retained the complete activity over the whole incubationtime, while the secreted and released enzyme lost about 90% of theactivity after an incubation of 65 hours. This indicates, that theimmobilization of the described enzyme into the cell wall of yeastprotects the enzyme against inactivation, presumably throughproteinases, and thereby increases the stability significantly. Furtherinsight into the localization of the different gene products wasobtained by Western analysis. Therefore, cells were harvested bycentrifugation and washed in 10 mM Tris.HCl, pH 7.8; 1 mM PMSF at 0° C.and all subsequent steps were performed at the same temperature. Threeml isolation buffer and 10 g of glass beads were added per gram of cells(wet weight). The mixture was shaken in a Griffin shaker at 50% of itsmaximum speed for 30 minutes. The supernatant was isolated and the glassbeads were washed with 1M NaCl and 1 mM PMSF until the washes wereclear. The supernatant and the washes were pooled. The cell walls wererecovered by centrifugation and were subsequently washed in 1 mM PMSF.

Non-covalently bound proteins or proteins bound through disulphidebridges were released from cell walls by boiling for 5 minutes in 50 mMTris.HCl, pH 7.8; containing 2% SDS, 100 mM EDTA and 40 mMβ-mercaptoethanol. The SDS-extracted cell walls were washed severaltimes in 1 mM PMSF to remove SDS. Ten mg of cell walls (wet weight) weretaken up in 20 l 100 mM sodium acetate, pH 5.0, containing 1 mM PMSF. Tothis, 0.5 mU of the β-1,3-glucanase (Laminarase; Sigma L5144) was usedas a source of β-1,3-glucanase) was added followed by incubation for 2hours at 37° C. Subsequently another 0.5 mU of β-1,3-glucanase wasadded, followed by incubation for another 2 hours at 37° C.

Proteins were denatured by boiling for 5 minutes preceding Endo-Htreatment. Two mg of protein were incubated in 1 ml 50 mM potassiumphosphate, pH 5.5, containing 100 mM β-mercaptoethanol and 0.5 mM PMSFwith 40 mU Endo-H (Boehringer) for 48 hours at 37° C. Subsequently 20 mUEndo-H were added followed by 24 hours of incubation at 37° C.

Proteins were separated by SDS-PAGE according to Laemmli (see reference13) in 2.2.-20% gradient gels. The gels were blotted by electrophoretictransfer onto Immobilon polyvinylidene-difluoride membrane (Millipore)as described by Towbin et al. (see reference 14). In case of highlyglycosylated proteins a subsequently mild periodate treatment wasperformed in 50 mM periodic acid, 100 mM sodium acetate, pH 4.5, forseveral hours at 4° C. All subsequent incubations were carried out atroom temperature. The blot was blocked in PBS, containing 0.5% gelatineand 0.5% Tween-20, for one hour followed by incubation for 1 hour inprobe buffer (PBS, 0.2% gelatine, 0.1% Tween-20) containing 1:200diluted serum. The blot was subsequently washed several times in washingbuffer (PBS; 0.2% gelatine; 0.5% Tween-20) followed by incubation for 1hour in probe-buffer containing ¹²⁵ I-labelled protein A (Amersham).After several washes in washing buffer, the blot was air-dried, wrappedin Saran (Dow) and exposed to X-omat S film (Kodak) with intensifyingscreen at -70° C. An Omnimedia 6cx scanner and the Adobe Photoshopprogramme were used to quantify the amount of labelled protein. Theresults of the various protein isolation procedures from bothtransformants are given in FIG. 5. While for the transformantscomprising the pSY13 plasmid the overall mass of the enzyme waslocalized in the medium, with only minor amounts of enzyme moreentrapped than bond in the cell wall (FIG. 5A)--which could completelybe removed by SDS extraction--the fusion protein was tightly bound tothe cell wall; with only small amounts of α-galactosidase/α-agglutinindelivered into the surrounding culture fluid or being SDS extractable.In contrast to the laminarinase extraction of cell walls from cellsexpressing the free α-galactosidase, where no further liberation of anymore enzyme was observed, identical treatment of fusion enzymeexpressing cells released the overall bulk of the enzyme. This indicatesthat the fusion protein is intimately associated with the cell wallglucan in S. cerevisiae, like α-agglutinin, while α-galactosidase aloneis not. The subsequently performed EndoH treatment showed a heavyglycosylation of the fusion protein, a result, entirely in agreementwith the described extended glycosylation of the C-terminal part ofα-agglutinin.

2. Localization

Immunofluorescent labelling with anti-α-galactosidase serum wasperformed on intact cells to determine the presence and distribution ofα-galactosidase/α-agglutinin fusion protein in the cell wall.Immunofluorescent labelling was carried out without fixing according toWatzele et al. (see reference 15). Cells of OD₅₃₀ =2 were isolated andwashed in TBS (10 mM Tris.HCl, pH 7.8, containing 140 mM NaCl, 5 mM EDTAand 20 μg/ml cycloheximide). The cells were incubated inTBS+anti-α-galactosidase serum for 1 hour, followed by several washingsin TBS. A subsequent incubation was carried out with FITC-conjugatedanti-rabbit IgG (Sigma) for 30 minutes. After washing in TBS, cells weretaken up in 10 mM Tris.HCl, pH 9.0, containing 1 mg/mlp-phenylenediamine and 0.1% azide and were photographed on a Zeiss 68000microscope. The results of these analysis are given in FIG. 6, showingclearly that the chimeric α-galactosidase/α-agglutinin is localized atthe surface of the yeast cell. Buds of various sizes, even very smallones very uniformly labelled, demonstrates that the fusion enzyme iscontinuously incorporated into the cell wall throughout the cell cycleand that it instantly becomes tightly linked.

3. Activity

To quantitatively assay α-galactosidase activity, 200 μl samplescontaining 0.1M sodium-acetate, pH 4.5 and 10 mMp-nitrophenyl-α-D-galactopyranoside (Sigma) were incubated at 37° C. forexactly 5 minutes. The reaction was stopped by addition of 1 ml 2%sodium carbonate. From intact cells and cell walls, removed bycentrifugation and isolated and washed as described, the α-galactosidaseactivity was calculated using the extinction coefficient ofp-nitrophenol of 18.4 cm² /μmole at 410 nm. One unit was defined as thehydrolysis of 1 μmole substrate per minute at 37° C.

                  TABLE 1                                                         ______________________________________                                        Distribution of free and immobilized α-galactosidase activity in         yeast cells                                                                                  α-Galactosidase activity                                                 (U/g F.W. cells)                                             Expressed       Growth    Intact Isolated                                       protein medium cells cell walls                                             ______________________________________                                        α-galactosidase                                                                         14.7      0.37   0.01                                           αGal/αAGG fusion protein 0.54 13.3 10.9                         ______________________________________                                         Transformed MT302/1C cells were in exponential phase (OD.sub.530 = 2). On     unit is defined as the hydrolysis of 1 μmole of                            pnitrophenyl-D-galactopyranoside per minute at 37° C.             

The results are summarized in Table 1. While the overall majority ofα-galactosidase was distributed in the culture fluid, most of the fusionproduct was associated with the cells, primarily with the cell wall.Taking together the results shown in FIGS. 3 to 6 and in Table 1, itcould be calculated that the enzymatic α-galactosidase activity of thechimeric enzyme is as good as that of the free enzyme. Moreover, duringstationary phase, the activity of the α-galactosidase in the growthmedium decreased, whereas the activity of the cell wall associatedα-galactosidase α-agglutinin fusion remained constant, indicating thatthe cell associated fusion protein is protected from inactivation orproteolytic degradation.

N.B. The essence of this EXAMPLE was published during the priority yearby M. P. Schreuder et al. (see reference 25).

EXAMPLE 2A Immobilized Humicola Lipase/α-Agglutinin on the Surface of S.cerevisiae. (inducible expression of immobilized enzyme system)

The construction and isolation of the 1.4 kb NheI/HindIII fragmentcontaining the C-terminal part of α-agglutinin has been described inEXAMPLE 1. Plasmid pUR7021 contains an 894 bp long syntheticallyproduced DNA fragment encoding the lipase of Humicola (see reference 16and SEQ ID NO: 7 and 8), cloned into the EcoRI/HindIII restriction sitesof the commercially available vector pTZ18R (see FIG. 7). For the properone-step modification of both the 5' end and the 3' end of the DNA partcoding for the mature lipase, the PCR technique can be applied.Therefore the DNA oligonucleotides lipol (see SEQ ID NO: 3) and lipo2(see SEQ ID NO: 6) can be used as primers in a standard PCR protocol,generating an 826 bp long DNA fragment with an EagI and a HindIIIrestriction site at the ends, which can be combined with the larger partof the EagI/HindIII digested pUR2650, a plasmid containing theα-galactosidase gene preceded by the invertase signal sequence asdescribed earlier in this specification, thereby generating plasmidpUR2970A (see FIG. 7).

PCR oligonucleotides for the in-frame linkage of Humicola lipase and theC-terminus of α agglutinin.

a: PCR oligonucleotides for the transition between SUC2 signal sequenceand the N-terminus of lipase.

                                 >mature lipase                                                           EagI   E   V   S   Q   D   L   F                        primer lipol: 5'-GGG GCG GCC GAG GTC TCG CAA GAT CTG GA-3'                                                ||| |.ve                                          rtline.| |.vertlin                                          e.| ||.ve                                          rtline. ||.vertlin                                          e. |||                                             ||                      lipase: 3'-TAA GCA GCT CTC CAG AGC GTT CTG GAC CTG TTT-5'                     (non-coding strand, see SEQ ID NO: 4)                                   

b: PCR oligonucleotides for the in frame transition between C-terminusof lipase and C-terminal part of α-agglutinin.

                          F   G   L   I   G   T   C   L                             lipase        5'-TTC GGG TTA ATT GGG ACA TGT CTT TAG TGC GA-3'                (cod. strand)        ||| ||                                                 | |.vertlin                                                 e.| |.vertl                                                 ine.| |.ver                                                 tline.| |.v                                                 ertline.| |                                                 ||                                                          ||.vertline                                                 . ||.vertli                                                 ne. ||                                                       primer            3'-CCC                                                     AAT TAA CCC TGT ACA GAA CGA                                                   TCG GAA TTC GAACCCC-5'                                                         lipo2:                                                                                              NheI                                                        HindIII                       (for the part of the lipase coding strand see SEQ ID NO: 5)             

Through the PCR method a NheI site will be created at the end of thecoding sequence of the lipase, allowing the in-frame linkage between theDNA coding for lipase and the DNA coding for the C-terminal part ofα-agglutinin. Plasmid pUR2970A can then be digested with NheI andHindIII and the 1.4 kb NheI/HindIII fragment containing the C-terminalpart of α-agglutinin from plasmid pUR2968 can be combined with thelarger part of NheI and HindIII treated plasmid pUR2970A, resulting inplasmid pUR2971A. From this plasmid the 2.2 kb EagI/HindIII fragment canbe isolated and ligated into the EagI- and HindIII-treated pUR2741,whereby plasmid pUR2741 is a derivative of pUR2740 (see reference 17),where the second EagI restriction site in the already inactive Tetresistance gene was deleted through NruI/SalI digestion. The SalI sitewas filled in prior to religation. The ligation then results in pUR2972Acontaining the GAL7 promoter, the invertase signal sequence, thechimeric lipase/α-agglutinin gene, the 2 μm sequence, the defective Leu2promoter and the Leu2 gene. This plasmid can be used for transforming S.cerevisiae and the transformed cells can be cultivated in YP mediumcontaining galactose as an inducer without repressing amounts of glucosebeing present, which causes the expression of the chimericlipase/α-agglutinin gene.

The expression, secretion, localization and activity of the chimericlipase/α-agglutinin can be analyzed using similar procedures as given inEXAMPLE 1.

In a similar way variants of Humicola lipase, obtained via rDNAtechniques, can be linked to the C-terminal part of α-agglutinin, whichvariants can have a higher stability during (inter)esterificationprocesses.

EXAMPLE 2B Immobilized Humicola Lipase/α-Agglutinin on the Surface of S.cerevisiase (inducible expression of immobilized enzyme system)

EXAMPLE 2A describes a protocol for preparing a particular construct.Before carrying out the work it was considered more convenient to usethe expression vector described in EXAMPLE 1, so that the constructionroute given in this EXAMPLE 2B differs on minor points from theconstruction route given in EXAMPLE 2A and the resulting plasmids arenot identical to those described in EXAMPLE 2A. However, the essentialgene construct comprising the promoter, signal sequence, and thestructural gene encoding the fusion protein are the same in EXAMPLES 2Aand 2B.

1. Construction

The construction and isolation of the 1.4 kb NheI/HindIII fragmentencoding the C-terminal part of α-agglutinin cell wall protein has beendescribed in EXAMPLE 1. The plasmid pUR7033 (resembling pUR7021 ofEXAMPLE 2A) was made by treating the commercially available vectorpTZ18R with EcoRI and HindIII and ligating the resulting vector fragmentwith an 894 bp long synthetically produced DNA EcoRI/HindIII fragmentencoding the lipase of Humicola (see SEQ ID NO: 7 and 8, and reference16).

For the fusion of the lipase to the C-terminal, cell wallanchor-comprising domain of α-agglutinin, plasmid pUR7033 was digestedwith EagI and HindIII, and the lipase coding sequence was isolated andligated into the EagI- and HindIII-digested yeast expression vector pSY1(see reference 27), thereby generating pUR7034 (see FIG. 13). This is a2 μm episomal expression vector, containing the α-galactosidase genedescribed in EXAMPLE 1, preceded by the invertase (SUC2) signal sequenceunder the control of the inducible GAL7 promoter.

Parallel to this digestion, pUR7033 was also digested with EcoRV andHindIII, thereby releasing a 57 bp long DNA fragment, possessing codonsfor the last 15 carboxyterminal amino acids. This fragment was exchangedagainst a small DNA fragment, generated through the hybridisation of thetwo chemically synthesized deoxyoligonucleotides SEQ ID NO: 9 and SEQ IDNO: 10. After annealing of both DNA strands, these two oligonucleotidesessentially reconstruct the rest of the 3' coding sequence of theinitial lipase gene, but additionally introduce downstream of the lipasegene a new NheI restriction site, followed by a HindIII site in closevicinity, whereby the first three nucleotides of the NheI site form thecodon for the last amino acid of the lipase. The resulting plasmid wasdesignated pUR2970B. Subsequently, this construction intermediate wasdigested with EagI and NheI, the lipase encoding fragment was isolated,and, together with the 1.4 kb NheI/HindIII fragment of pUR2968 ligatedinto the EagI- and HindIII-cut pSY1 vector. The outcome of this3-point-ligation was called pUR2972B (see FIG. 14), the finallipolase-α-agglutinin yeast expression vector.

This plasmid was used for transforming S. cerevisiae strain SU10 asdescribed in reference 17 and the transformed cells were cultivated inYP medium containing galactose as the inducer without repressing amountsof glucose being present, which causes the expression of the chimericlipase/α-agglutinin gene.

2. Activity

To quantify the lipase activity, two activity measurements with twoseparate substrates were performed. In both cases, SU10 yeast cellstransformed with either plasmid pUR7034 or pSY1 served as control.Therefore, yeast cell transformants containing either plasmid pSY1 orplasmid pUR7034 or plasmid pUR2972B were grown up for 24 h inYNB-glucose medium supplied with histidine and uracil, then diluted 1:10in YP-medium supplied with 5% galactose, and again cultured. After 24 hincubation at 30° C., a first measurement for both assays was performed.

The first assay applied was the pH stat method. Within this assay, oneunit of lipase activity is defined as the amount of enzyme capable ofliberating one micromole of fatty acid per minute from a triglyceridesubstrate under standard assay conditions (30 ml assay solutioncontaining 38 mM olive oil, considered as pure trioleate, emulsifiedwith 1:1 w/w gum arabic, 20 mM calcium chloride, 40 mM sodium chloride,5 mM Tris, pH 9.0, 30° C.) in a radiometer pH stat apparatus (pHM 84 pHmeter, ABU 80 autoburette, TTA 60 titration assembly). The fatty acidsformed were titrated with 0.05N NaOH and the activity measured was basedon alkali consumption in the interval between 1 and 2 minutes afteraddition of putative enzyme batch. To test for immobilized lipaseactivity, 1 ml of each culture was centrifuged, the supernatant wassaved, the pellet was resuspended and washed in 1 ml 1M sorbitol,subsequently again centrifuged and resuspended in 200 μl 1M sorbitol.From each type of yeast cell the first supernatant and the washed cellswere tested for lipase activity.

A: Lipase activity after 24 h (LU/ml)

    ______________________________________                                                      cell bound                                                                           culture fluid                                            ______________________________________                                        pSY1            5.9      8.8                                                    pUR7034 24.1 632.0                                                            pUR2972B-(1) 18.7 59.6                                                        pUR2972B-(2) 24.6 40.5                                                      ______________________________________                                    

B: Lipase activity after 48 h (LU/ml)

    ______________________________________                                                 cell bound                                                                              culture fluid                                                                           OD660                                            ______________________________________                                        pSY1       6.4         4.3       .sup.- 40                                      pUR7034 215.0 2750.0 .sup.- 40                                                pUR2972B-(1) 37.0 87.0 .sup.- 40                                              pUR2972B-(2) 34.0 82.0 .sup.- 40                                            ______________________________________                                    

The rest of the yeast cultures was further incubated, and essentiallythe same separation procedure was done after 48 hours. Dependent on theinitial activity measured, the actual volume of the sample measureddeviated between 25 μl and 150 μl.

This series of measurements indicates, that yeast cells comprising theplasmid coding for the lipase-α-agglutinin fusion protein in factexpress some lipase activity which is associated with the yeast cell.

An additional second assay was performed to further confirm theimmobilization of activity of lipase on the yeast cell surface. Briefly,within this assay, the kinetics of the PNP (=paranitrophenyl) releasefrom PNP-butyrate is determined by measurement of the OD at 400 nm.Therefore, 10 ml cultures containing yeast cells with either pSY1,pUR7034 or pUR2972B were centrifuged, the pellet was resuspended in 4 mlof buffer A (0.1M NaOAc, pH 5.0 and 1 mM PMSF), from this 4 ml 500 μlwas centrifuged again and resuspended in 500 μl PNB-buffer (20 mMTris-HCl, pH 9.0, 20 mM CaCl2, 25 mM NaCl), centrifuged once again, andfinally resuspended in 400 μl PNB buffer. This fraction was used todetermine the cell bound fraction of lipase.

The remaining 3500 μl were spun down, the pellet was resuspended in 4 mlA, to each of this, 40 μl laminarinase (ex mollusc, 1.25 mU/μl) wasadded and first incubated for 3 hours at 37° C., followed by anovernight incubation at 20° C. Then the reaction mixture, stillcontaining intact cells, were centrifuged again and the supernatant wasused to determined the amount of originally cell wall bound materialreleased through laminarinase incubation. The final pellet wasresuspended in 400 μl PNP buffer, to calculate the still cell associatedpart. The blank reaction of a defined volume of specific culturefraction in 4 ml assay buffer was determined, and than the reaction wasstarted through addition of 80 μl of substrate solution (100 mMPNP-butyrate in methanol), and the reaction was observed at 25° C. at400 nm in a spectrophotometer.

    __________________________________________________________________________           cell bound                                                                           activity in                                                                         laminarinase                                                                         laminarinase                                         activity* the medium extract extracted cells OD660                          __________________________________________________________________________    pSY1   0.001 (116 μl)                                                                    0.001 0.028  0.000  2.6                                           pUR7034 0.293 (220 μl) 0.446 0.076 0.985 2.36                              pUR2972B-(1) 0.494 (143 μl) 0.021 0.170 0.208 2.10                       __________________________________________________________________________     *unless otherwise mentioned, the volume of enzyme solution added was 20       μl                                                                    

This result positively demonstrates that a significant amount of lipaseactivity is immobilized on the surface yeast cell, containing plasmidpUR2972B. Here again, incorporation took place in such a way, that thereaction was catalyzed by cell wall inserted lipase of intact cells,indicated into the exterior orientated immobilization. Furthermore, therelease of a significant amount of lipase activity after incubation withlaminarinase again demonstrates the presumably covalent incorporation ofa heterologous enzyme through gene fusion with the C-terminal part ofα-agglutinin.

3. Localization

The expression, secretion, and subsequent incorporation of thelipase-α-agglutinin fusion protein into the yeast cell wall was alsoconfirmed through Immunofluorescent labelling with anti-lipolase serumessentially as described in EXAMPLE 1, item 2. Localization.

As can be seen in FIG. 15, the Immunofluorescent stain shows essentiallyan analogous picture as the α-galactosidase immuno stain, with clearlydetectable reactivity on the outside of the cell surface (see FIG. 15 Ashowing a clear halo around the cells and FIG. B showing a lightercircle at the surface of the cells), but neither in the medium nor inthe interior of the cells. Yeast cells expressing pUR2972B, the Humicolalipase-α-agglutinin fusion protein, become homogeneously stained on thesurface, indicating the virtually entire irmnobiLization of a chimericenzyme with an α-agglutinin C-terminus on the exterior of a yeast cell.In the performed control experiment SU10 yeast cells containing plasmidpUR7034 served as a control and here, no cell surface bound reactivityagainst the applied anti-lipase serum could be detected.

In a similar way variants of Humicola lipase, obtained via rDNAtechniques, can be linked to the C-terminal part of α-agglutinin, whichvariants can have a higher stability during (inter)esterificationprocesses.

EXAMPLE 3 Immobilized Humicola Lipase/α-Agglutinin on the Surface of S.cerevisiae (constitutive expression of immobilized enzyme system)

Plasmid pUR2972 as described in EXAMPLE 2 can be treated with EagI andHindIII and the about 2.2 kb fragment containing the lipase/α-agglutiningene can be isolated. Plasmid pSY16 can be restricted with EagI andHindIII and between these sites the 2.2 kb fragment containing thelipase/α-agglutinin fragment can be ligated resulting in pUR2973. Thepart of this plasmid that is involved in the production of the chimericenzyme is similar to pUR2972 with the exception of the signal sequence.Whereas pUR2972 contains the SUC2-invertase-signal sequence, pUR2973contains the α-mating factor signal sequence (see reference 18).Moreover the plasmid pUR2973 contains the Leu2 marker gene with thecomplete promoter sequence, instead of the truncated promoter version ofpUR2972.

EXAMPLE 4 Immobilized Geotrichum Lipase/α-Agglutinin on the Surface ofS. cerevisiae

The construction and isolation of the 1.4 kb NheI/HindIII fragmentcomprising the C-terminal part of AGα-1 (α-agglutinin) gene has beendescribed in EXAMPLE 1. For the in-frame gene fusion of the DNA codingfor the C-terminal membrane anchor of α-agglutinin to the completecoding sequence of Geotichum candidum lipase B from strain CMICC 335426(see FIG. 8 and SEQ ID NO: 11 and 12), the plasmid pUR2974 can be used.This plasmid, derived from the commercially available pBluescript II SKplasmid, contains the cDNA coding for the complete G. candidum lipase IIon an 1850 bp long EcoRI/XhoI insert (see FIG. 9).

To develop an expression vector for S. cerevisiae with homologous signalsequences, the N-terminus of the mature lipase B was determinedexperimentally by standard techniques. The obtained amino acid sequenceof "Gln-Ala-Pro-Thr-Ala-Val . . ." is in complete agreement with thecleavage site of the signal peptidase on the G. candidum lipase II (seereference 19).

For the fusion of the mature lipase B to the S. cerevisiae signalsequences of SUC2 (invertase) or α-mating factor (prepro-αMF) on onehand and the in-frame fusion to the 3' part of the AGα1 gene PCRtechnique can be used. The PCR primer lipo3 (see SEQ ID NO: 13) can beconstructed in such a way, that the originally present EagI site in the5'-part of the coding sequence (spanning codons 5-7 of the matureprotein) will become inactivated without any alteration in the aminoacid sequence. To facilitate the subsequent cloning procedures, the PCRprimer can further contain a new EagI site at the 5' end, for thein-frame ligation to SUC2 signal sequence or prepro-αMF sequence,respectively. The corresponding PCR primer lipo4 (see SEQ ID NO: 16)contains an extra NheI site behind the nucleotides coding for theC-terminus of lipase B, to ensure the proper fusion to the C-terminalpart of α-agglutinin.

PCR oligonucleotides for the in frame linkage of G. candidum lipase IIto the SUC2 signal sequence and the C-terminal part of α-agglutinin.

a: N-terminal transition to either prepro αMF sequence or SUC2 signalsequence.

                     EagI   A   Q   A   P   R   P   S   L   N                       primer lipo3: 5'-GGG GCG GCC GCG CAG GCC CCA AGG CGG TCT CTC AAT-3'                                                                   |.vertl                                             ine.  ||.vertli                                             ne. ||.vertline                                             . |||                                               || |.                                             vertline.| |.ve                                             rtline.| |.vert                                             line.| |.vertli                                             ne.|                          lipaseII:         3'-GAC CGG GTC CGG GGT GCC GCC AGA GAG TTA-5'             (non-cod.  strand, see SEQ ID NO: 14) )                                   

b: C-terminal fusion to C part of α-agglutinin

               S   N   F   E   T   D   V   N   L   Y   G                            lipase: 5'-CA AAC TTT GAG ACT GAC GTT AAT CTC TAC GGT TAA AAC-3'                                                          (cod.  strand)                                                                  | |.vert                                                 line.| |.ve                                                 rtline.| |.                                                 vertline.| .vertline                                                 .||                                                         ||.vertline                                                 . ||.vertli                                                 ne. ||.vert                                                 line. ||.ve                                                 rtline. ||.                                                 vertline.                          primer lipo4:              3'-C TGA CTG CAA TTA GAG ATG CCA CGATCG                                                       CCCC-5'                                                                            NheI                        (for the part of the lipase coding strand see SEQ ID NO: 15)              

The PCR product with the modified ends can be generated by standard PCRprotocols, using instead of the normal Ampli-Taq polymerase the newthermostable VENT polymerase, which also exhibits proofreading activity,to ensure an error-free DNA template. Through digestion of the formerlydescribed plasmid pUR2972 with EagI (complete) and NheI (partial), theHumicola lipase fragment can be exchanged against the DNA fragmentcoding for lipase B, thereby generating the final S. cerevisiaeexpression vector pUR2975 (see FIG. 9).

The Humicola lipase-α-agglutinin fusion protein coding sequence can beexchanged against the lipase B/α-agglutinin fusion construct describedabove by digestion of the described vector pUR2973 with EagI/HindIII,resulting in pUR2976 (see FIG. 9).

EXAMPLE 5 Immobilized Rhizomucor miehei Lipase/α-Agglutinin on theSurface of S. cerevisiae

The construction and isolation of the 1.4 kb NheI/HindIII fragmentencoding the C-terminal part of α-agglutinin has been described inEXAMPLE 1. The plasmid pUR2980 contains a 1.25 kb cDNA fragment clonedinto the SmaI site of commercially available pUC18, which (syntheticallysynthesizable) fragment encodes the complete coding sequence oftriglyceride lipase of Rhizomucor miehei (see reference 20), an enzymeused in a number of processes to interesterify triacylglycerols (seereference 21) or to prepare biosurfactants (see reference 22). Besidethe 269 codons of the mature lipase molecule, the fragment also harbourscodons for the 24 amino acid signal peptide as well as 70 amino acids ofthe propeptide. PCR can easily be applied to ensure the proper fusion ofthe gene fragment encoding the mature lipase to the SUC2 signal sequenceor the prepro α-mating factor sequence of S. cerevisiae, as well as thein-frame fusion to the described NheI/HindIII fragment. The followingtwo primers, lipo5 (see SEQ ID NO: 17) and lipo6 (see SEQ ID NO: 20),will generate a 833 bp DNA fragment, which after Proteinase K treatmentand digestion with EagI and NheI can be cloned as an 816 bp longfragment into the EagI/NheI digested plasmids pUR2972 and pUR2973,respectively (see FIG. 7).

                             EagI   A   S   I   D   G   G   I                       lipo5: 5'-CCC GCG GCC GCG AGC ATT GAT GGT GGT ATC-3'                                              ||| ||.                                                   vertline. |.vertli                                                   ne.| |.ve                                                   rtline.| .vertline                                                   .||                                                         ||.vertli                                                   ne.                              lipase (non-cod. strand):                 3'-TCG TAA CTA GCA CCA TAG-5'     (for the part of the lipase non-coding strand, see SEQ ID NO: 18)               -                                                                                                  N   T   G   L   C   T                                                                                  lipase (cod.                                                                  strand):           5'-AAC                                                    ACA GGC CTC TGT ACT-3'                                                                       |.v                                                   ertline.|                                                            ||.vertli                                                   ne. ||.ve                                                   rtline. |.vertline                                                   .| |.vert                                                   line.| |.                                                   vertline.|                                                            Lipo6:           3'-TTG                                                      TGT CCG GAG ACA TGA                                                           CGATCGCGCC-5'-                                                                NheI                           (for the part of the lipase coding strand, see SEQ ID NO: 19)             

These new S. cerevisiae expression plasmids contain the GAL7 promoter,the invertase signal sequence (pUR2981) or the prepro-α-mating factorsequence (pUR2982), the chimeric Rhizomucor miehei lipase/α-agglutiningene, the 2 μm sequence, the defective (truncated) Leu2 promoter and theLeu2 gene. These plasmids can be transformed into S. cerevisiae andgrown and analyzed using protocols described in earlier EXAMPLES.

EXAMPLE 6 Immobilized Aspergillus niger Glucose Oxidase/GPI AnchoredCell Wall Proteins on the Surface of S. cerevisiae

Glucose oxidase (β-D:oxygen 1-oxidoreductase, EC 1.1.3.4) fromAspergillus niger catalyses the oxidation of β-D-glucose toglucono-δ-lactone and the concomitant reduction of molecular oxygen tohydrogen peroxide. The fungal enzyme consists of a homodimer ofmolecular weight 150,000 containing two tightly bound FAD co-factors.Beside the use in glucose detection kits the enzyme is useful as asource of hydrogen peroxide in food preservation. The gene was clonedfrom both cDNA and genomic libraries, the single open reading framecontains no intervening sequences and encodes a protein of 605 aminoacids (see reference 23).

With the help of two proper oligonucleotides the coding part of thesequence is adjusted in a one-step modifying procedure by PCR in such away that a fusion gene product will be obtained coding for glucoseoxidase and the C-terminal cell wall anchor of the FLO1 gene product orα-agglutinin. Thus, some of the plasmids described in former EXAMPLEScan be utilized to integrate the corresponding sequence in-frame betweenone of the signal sequences used in the EXAMPLES and the NheI/HindIIIpart of the AGα1 gene.

Since dimerisation of the two monomers might be a prerequisite foractivity, in an alternative approach the complete coding sequence forglucose oxidase without the GPI anchor can be expressed in S. cerevisiaetransformant which already contains the fusion construct. This can befulfilled by constitutive expression of the fusion construct containingthe GPI anchor with the help of the GAPDH or PGK promoter for example.The unbound not-anchored monomer can be produced by using a DNAconstruct comprising an inducible promoter, as for instance the GAL7promoter.

EXAMPLE 7 Process to Convert Raffinose, Stachyose and Similar Sugars inSoy Extracts with α-Galactosidase/α-Agglutinin Immobilized on Yeasts

The yeast transformed with plasmid pUR2969 can be cultivated on largescale. At regular intervals during cultivation the washed cells shouldbe analyzed on the presence of α-galactosidase activity on their surfacewith methods described in EXAMPLE 1. When both cell density andα-galactosidase activity/biomass reach their maximum, the yeast cellscan then be collected by centrifugation and washed. The washed cells canthen be added to soy extracts. The final concentration of the yeastcells can vary between 0.1 and 10 g/l, preferably the concentrationshould be above 1 g/l The temperature of the soy extract should be <8°C. to reduce the metabolic activity of the yeast cells. The conversionof raffinose and stachyose can be analyzed with HPLC methods and after95% conversion of these sugars the yeasts cells can be removed bycentrifugation and their α-galactosidase activity/g biomass can bemeasured. Centrifugates with a good activity can be used in a subsequentconversion process, whereas centrifugates with an activity of less then50% of the original activity can be resuscitated in the growth mediumand the cells can be allowed to recover for 2 to 4 hours. Thereafter thecells can be centrifuged, washed and subsequently be used in asubsequent conversion process.

EXAMPLE 8 Production of Biosurfactants Using HumicolaLipase/α-Agglutinin Immobilized on Yeasts.

The yeast transformed with plasmid pUR2972 or pUR2973 can be cultivatedon large scale. At regular intervals during cultivation the washed cellscan be analyzed on the presence of lipase activity on their surface withmethods described in EXAMPLE 2. When both cell density andlipase/biomass reache their maximum, the yeast cells can be collected bycentrifugation and washed. The washed cells can be suspended in a smallamount of water and added to a reactor tank containing a mix of fattyacids, preferably of a chain length between 12-18 carbon atoms andsugars, preferably glucose, galactose or sucrose. The totalconcentration of the water (excluding the water in the yeast cells)might be below 0.1%. The final concentration of the yeast cells can varybetween 0.1 and 10 g/l, preferably the concentration is above 1 g/l. Thetank has to be kept under an atmosphere of N₂ and CO₂ in order to avoidoxidation of the (unsaturated) fatty acids and to minimize the metabolicactivity of the yeasts. The temperature of mixture in the tank should bebetween 30-60° C., depending on type of fatty acid used. The conversionof fatty acids can be analyzed with GLC methods and after 95% conversionof these fatty acids the yeasts cells can be removed by centrifugationand their lipase activity/g biomass can be measured. Centrifugates witha good activity can be used in a subsequent conversion process, whereascentrifugates with an activity of less then 50% of the original activitycan be resuscitated in the growth medium and the cells can be allowed torecover for 2 to 8 hours. Thereafter the cells can be centrifuged again,washed and used in a subsequent conversion process.

EXAMPLE 9 Production of Special Types of Triacylglycerols usingRhizomucor miehei Lipase/α-Agglutinin Immobilized on Yeasts.

The yeast transformed with plasmid pUR2981 or pUR2982 can be cultivatedon a large scale. At regular intervals during cultivation the washedcells can be analyzed on the presence of lipase activity on theirsurface with methods described in EXAMPLE 1. When both cell density andlipase/biomass reach their maximum, the yeast cells can be collected bycentrifugation and washed. The washed cells can be suspended in a smallamount of water and can be added to a reactor tank containing a mix ofvarious triacylglycerols and fatty acids. The total concentration of thewater (excluding the water in the yeast cells) might be below 0.1%. Thefinal concentration of the yeast cells can vary between 0.1 and 10 g/l,preferably the concentration is above 1 g/l. The tank has to be keptunder an atmosphere of N₂ and CO₂ in order to avoid oxidation of the(unsaturated) fatty acids and to minimize the metabolic activity of theyeasts. The temperature of mixture in the tank should be between 30-70°C., depending on types of triacylglycerol and fatty acid used. Thedegree of interesterification can be analyzed with GLC/MS methods andafter formation of at least 80% of the theoretical value of the desiredtype of triacylglycerol the yeasts cells can be removed bycentrifugation and their lipase activity/g biomass can be measured.Centrifugates with a good activity can be used in a subsequentconversion process, whereas centrifugates with an activity of less then50% of the original activity is resuscitated in the growth medium andthe cells should be allowed to recover 2 to 8 hours. After that thecells can be centrifuged, washed and used in a subsequentinteresterification process.

Baker's yeasts of strain MT302/1C, transformed with either plasmid pSY13or plasmid pUR2969 (described in EXAMPLE 1) were deposited under theBudapest Treaty at the Centraalbureau voor Schimmelcultures (CBS) onJul. 3, 1992 under provisional numbers 330.92 and 329.92, respectively.

EXAMPLE 10 Immobilized Humicola Lipase/FLO1 Fusion on the Surface of S.cerevisiae

Flocculation, defined as "the (reversible) aggregation of dispersedyeast cells into flocs" (see reference 24), is the most importantfeature of yeast strains in industrial fermentations. Beside this it isof principal interest, because it is a property associated with cellwall proteins and it is a quantitative characteristic. One of the genesassociated with the flocculation phenotype in S. cerevisiae is the FLO1gene. The gene is located at approximately 24 kb from the right end ofchromosome I and the DNA sequence of a clone containing major parts ofFLO1 gene has very recently been determined (see reference 26). Thesequence is given in FIG. 11 and SEQ ID NO: 21 and 22. The clonedfragment appeared to be approximately 2 kb shorter than the genomic copyas judged from Southern and Northern hybridizations, but encloses bothends of the FLO1 gene. Analysis of the DNA sequence data indicates thatthe putative protein contains at the N-terminus a hydrophobic regionwhich confirms a signal sequence for secretion, a hydrophobic C-terminusthat might function as a signal for the attachment of a GPI-anchor andmany glycosylation sites, especially in the C-terminus, with 46.6%serine and threonine in the arbitrarily defined C-terminus (aa 271-894).Hence, it is likely that the FLO1 gene product is localized in anorientated fashion in the yeast cell wall and may be directly involvedin the process of interaction with neighbouring cells. The cloned FLO1sequence might therefore be suitable for the immobilization of proteinsor peptides on the cell surface by a different type of cell wall anchor.

Recombinant DNA constructs can be obtained, for example by utilizing theDNA coding for amino acids 271-894 of the FLO1 gene product, i.e.polynucleotide 811-2682 of FIG. 11. Through application of two PCRprimers pcrflo1 (see SEQ ID NO: 23) and pcrflo2 (see SEQ ID NO: 26) NheIand HindIII sites can be introduced at both ends of the DNA fragment. Ina second step, the 1.4 kb NheI/HindIII fragment present in pUR2972(either A or B) containing the C-terminal part of α-agglutinin can bereplaced by the 1.9 kb DNA fragment coding for the C-terminal part ofthe FLO1 protein, resulting in plasmid pUR2990 (see FIG. 12), comprisinga DNA sequence encoding (a) the invertase signal sequence (SUC2)preceding (b) the fusion protein consisting of (b.1) the lipase ofHumicola (see reference 16) followed by (b.2) the C-terminus of FLO1protein (aa 271-894).

PCR oligonucleotides for the in frame connection of the genes encodingthe Humicola lipase and the C-terminal part of the FLO1 gene product.

                                   S   N   Y   A   V   S   T                        primer pcrflol 5'- GAATTC GCT AGC AAT TAT GCT GTC AGT AAC - 3'                            NheI    ||| ||.                                                     vertline. |.vert                                                     line.| .vertline                                                     .||                                                         ||.vert                                                     line. |.vertline                                                     .|                    FLO1 gene (non-coding strand)            3'- AGT TTA ATA CGA CAG TCA                                                         TGG TGA - 5'                 (for the part of the non-coding strand, see SEQ ID NO: 24)                       -                                                                          FLO1 coding strand                                                                              5'-AATAA AATTCGCGTTCTTTTTACG - 3'                                       ||||||.v                      ertline.||||.vertlin                      e.|||||.ver                      tline.|                                              primer pcrflo2:       3'-TTAAGCGCAAGAAAAATGC TTCGAACTCGAG - 5'                                         HindIII                                            (for the part of the coding strand, see SEQ ID NO: 25)                    

Plasmid pUR2972 (either A or B) can be restricted with NheI (partial)and HindIII and the NheI/HindIII fragment comprising the vector backboneand the lipase gene can be ligated to the correspondingly digested PCRproduct of the plasmid containing the FLO1 sequence, resulting inplasmid pUR2990, containing the GAL7 promoter, the S. cerevisiaeinvertase signal sequence, the chimeric lipase/FLO1 gene, the yeast 2 μmsequence, the defective Leu2 promoter and the Leu2 gene. This plasmidcan be transformed into S. cerevisiae and the transformed cells can becultivated in YP medium including galactose as inductor.

The expression, secretion, localization and activity of the chimericlipase/FLO1 protein can be analyzed using similar procedures as given inExample 1.

LITERATURE REFERENCES:

1. Monsan, P., Combes, D. (1988) "Enzyme stabilization byimmobilization"; in Meth. in Enzymol. Vol 137 584-598.

2. Kok, J. (1990) "Genetics of proteolytic systems of lactic acidsbacteria" FEMS Microbiol. Rev. 87 15-54.

3. Conzelmann, A., Fankhauser, C., Desponds, C. (1990) "Myoinositol getsincorporated into numerous membrane glycoproteins of S. cerevisiae:Incorporation is dependent on phosphomannomutase" (SEC53). EMBO 9653-661.

4. Lipke, P., N., Wojciechowicz, D., Kurjan, J. (1989) "AGα1 is thestructural gene for the Saccharomyces cerevisiae α-agglutinin, a cellsurface glycoprotein involved in cell-cell interactions during mating"Mol. Cell. Biol. 9 3155-3165.

5. Roy, A., Lu, C. F., Marykwas, D., Lipke, P., Kurja, J. (1991) "TheAGA1 gene product is involved in cell surface attachment of the S.cerevisiae cell adhesion glycoprotein a-agglutinin", Mol. Cell. Biol. 114196-4206.

6. Teunissen, A. W. R. H., van den Berg, J. A., Steensma, H. Y. (1993)"Physical localization of the flocculation gene FLO1 on chromosome I ofS. cerevisiae, Yeast 9 (1) 1-10.

7. Yanisch Perron, C., Viera, J., Messing, J. (1985) "Improved M13 phagecloning vectors and host strains: nucleotide sequence of the M13 mp18and pUC19 vectors." Gene 33 103-119.

8. Chung, C. T., Niemela, S. L., Miller, R. H. (1989) "One steppreparation of competent E. coli: Transformation and storage ofbacterial cells in the same solution" Proc. Natl. Acad. Sci. USA 862172-2175.

9. Sanger, F., Nicklen, S., Coulson, A. R. (1977) "DNA sequencing withchain terminating inhibitors" Proc. Natl. Acad. Sci. USA 74 5463-5467.

10. Hsiao, K. (1991) "A fast and simple procedure for sequencing doublestranded DNA with Sequenase" Nucl. Acids Res. 19 2787.

11. Klebe, R. J. J., Harriss, V., Sharp, Z. D., Douglas, M. G. (1983) "Ageneral method for polyethylene glycol induces genetic transformation ofbacteria and yeast" Gene 25 333-341.

12. Overbeeke, N., Fellinger, A. J., Toonen, M. Y., van Wassenaar, P.D., Verrips, C. T. (1989) "Cloning and nucleotide sequence of theα-galactosidase gene from Cyamopsis tetragonoloba" Plant Mol. Biol. 13541-550.

13. Laemmli, U. K. (1970) "Cleavage of structural proteins during theassembly of heads of bacteriophage T4." Nature 227 680-685.

14. Towbin, H. Steahelin, T., Gordon, J. (1979) "Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulose sheets:Procedure and some applications" Proc. Natl. Acad. Sci. USA 764350-4354.

15. Watzele, M., Klis, F., Tanner, W. (1988) "The immunological andmolecular characterization of α-agglutinin from S. cerevisiae" EMBO J. 71483-1488.

16. Boel. E., Huge-Jensen, B., Brown, J. D. (1989) "Humicola lipase andprocess for the production of recombinant Humicola lipases" EP-A1-0 305216.

17. Verbakel, J. M. A. (1991) "Heterologous gene expression in the yeastSaccharomyces cerevisiae" PhD thesis, Rijksuniversiteit Utrecht, TheNetherlands

18. Kurjan, J., Herskowitz, I. (1982) "Structure of a yeast PheromoneGene (MFα): A putative α-Factor precursor contains four tandem copiersof mature α-factor" Cell 30 933-943.

19. Shimada, Y., Sugihara, A., Tominaga, Y., Iizumi, T., Tsunasawa, S.(1989) "cDNA molecular cloning of Geotrichum candidum lipase" J.Biochem. 106 383-388.

20. Boel, E., Huge-Jensen, B., Christensen, M., Thim, L., Fiil, N.(1988) "Rhizomucor miehei Triglyceride Lipase is Synthesized as aPrecursor" Lipids, Vol.23, No 7, 701-706.

21. Schuch, R., Mukherjee, K. D. (1988) in "World conference onBiotechnology for the fat and oil industry" ISBN 0-935315-21-7, 328-329.

22. Kosaric, N., Cairus, W. L., Gray, N. C. C. (editors) (1987)"Biosurfactants and Biotechnology" Marcel Dekker Inc., New York, Vol.25.

23. Frederick, K. R., Tung, J., Emerik, R. S., Masiarz, F. R.,Chamberlain, S. H., Vasavada, A., Rosenberg, S. (1990) "Glucose oxidasefrom Aspergillus niger". J. Biol. Chem., Vol.265, No.7, 3793-3802.

24. Johnston, J. R., Reader, H. P. (1983) "Genetic control offlocculation" in `Yeast Genetics, Fundamental and applied aspects`,Spencer, J. F. T. (Editor), ISBN 0-540-90793-9, p. 205-224.

25. Schreuder, M. P., Brekelmans, S., Van den Ende, H., Klis, F. M.(1993) "Targeting of a Heterologous Protein to the Cell Wall ofSaccharomyces cerevisiae" Yeast 9 399-409.

26. Teunissen, A. W. R. H., Holub, E., Van der Hucht, J., Van Den Berg,J. A., Steensma, H. Y. (1993) "Sequence of the Open reading frame of theFLO1 Gene from Saccharomyces cerevisiae" YEAST 9 423-427.

27. Harmsen, M. M., Langedijk, A. C., Van Tuinen, E., Geerse, R. H.;Raue, H. A., Maat, J. (1993) Effect of a pmr1 disruption and differentsignal sequences on the intracellular processing and secretion ofCyamopsis tetragonoloba α-galactosidase by Saccharomyces cerevisiae Gene125 115-123.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 26                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6057 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Saccharomyce - #s cerevisiae                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 3653..5605                                                      (D) OTHER INFORMATION: - #/function= "sexual agglutinisation"                     /product=- # "alpha-agglutinin"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - AAGCTTTAGG TAAGGGAGGC AGGGGGAAAA GATACTGAAA TGACGGAAAA CG -             #AGAATATG     60                                                                 - - GAGCAGGGAG CAACTTTTAG AGCTTTACCC GTTAAAAGGT CAAATCGAGG CT -            #TCCTGCCT    120                                                                 - - TTGTCTGATT TTAGTAGTAC CGGAAGGTTT ATTACGCCCA AGAACAGTGC TT -            #GAATTGAG    180                                                                 - - TTCTCGGGAC ACGGGAAAGA CAATGGAAGA AAAATTTACA TTCAGTAGCC TT -            #ATATATGA    240                                                                 - - AATGCTGCCA AGCCACGTCT TTATAAGTAG ATAATGTCCC ATGAGCTGAA CT -            #ATGGGAAT    300                                                                 - - TTATGACGCA GTTCATTGTA TATATATTAC ATTAACTCTT TAGTTTAACA TC -            #TGAATTGT    360                                                                 - - TTTATAAAAT AACTTTTTGA ATTTTTTTAT GATCGCTTAG TTAAGTCTAT TA -            #TATCAGGT    420                                                                 - - TTTTTCATTC ATCATAATTG TTCGTTAAAT ATGAGTATAT TTAAATACAG GA -            #ATTAGTAT    480                                                                 - - CATTTGCAGT CACGAAAAGG GCCGTTTCAT AGAGAGTTTT CTTAATAAAG TT -            #GAGGGTTT    540                                                                 - - CCGTGATAGT TTTGAGGGGT TGTTTGAACT AGATTTACGC TTACCTTTCA AC -            #TGATTAAT    600                                                                 - - TTTTTCAGCG GGCTTATCAT AATCATCCAT CATAGCAGTC TTTCTGGACT TC -            #GTCGAGGA    660                                                                 - - CTGGCTTTCT GAATTTTGAC GGTCCCTATT AGCTCCAGTT GGAGGAATTG AG -            #TTACCTAC    720                                                                 - - AACTGGCAAG AGGTCTTTGT TTGGATTCAA AATAGGACTT TGTGGTAGCA GT -            #TTGGTTTT    780                                                                 - - ATTCAATCTA AAGATATGAG AAACAGGTTT TAAGTAAATC GATACTATTG TA -            #CCAATGTT    840                                                                 - - TAGCTCCAAT TCCTCCAAAA CGGTGGGATC TAATTTTGTG TTCATTTCTA TT -            #AGTGGCAA    900                                                                 - - CTCTCCGTCC AGTACTGATT TTAAAGATTC AAAAGTTATC GCGTTTGATA TA -            #CGAGACGT    960                                                                 - - TTTCGTTAAT GACAGCAATC TCCAATACAT CAGTGTTTTA TCTCTTAAGT CA -            #GGATTATT   1020                                                                 - - TTCGTGATCG GTGCATCCTT TTAATAAATC CATACAAAGT TCTTCAGTTT CC -            #TTTGTAGG   1080                                                                 - - ATTTCTGATG AAGAATTTTA TTGCTGAGTT CAGAATGGAA AATTGCACTT CT -            #AGCGTCTC   1140                                                                 - - ATTAAACATG TTTGAGGAAA AAACTCTAAA TAACTCCAGG TAGTTTGGAA TT -            #ACATCCGA   1200                                                                 - - ATATTGCGTT ATTATCCAGA TCATAGCGTT TTTTGATTCA GGTTCCTGTA CA -            #ACTTCAGT   1260                                                                 - - GTGTTTGACT AGTTCTGTTA CGTTTGCTTT AAAATTATTG GGATATTTCC TC -            #AAAATATT   1320                                                                 - - TCTGAAAACC GAAATAATCT CCTGGACGAC ATAATCAACA CCGAATTCTA AC -            #AAATCTAG   1380                                                                 - - TAGCACAGCG ACACAATCGT GTACAGAGTC TTCATCTAGC TTAACAGCGA GA -            #TTACCAAT   1440                                                                 - - GGCTCTGACT GATTTCCTTG ACATTTGAAT ATCAATATCT GTAGCATATT GT -            #TCCAACTC   1500                                                                 - - TTCTAGAATT CTTGGTAATG TTTCCTTGTT AGCTAAAAGA TATAAACACT CT -            #AATTTCGT   1560                                                                 - - GTCTTTGATG TATATGGGGT CATTGTACTC GATGAAAAAA TACGAAATGT CT -            #AGCCTGAG   1620                                                                 - - TAGAGATGAC TCCCTACTCA ATAAAAGAAG AATAACGTTT CTTAATACTA AA -            #AATTGTAA   1680                                                                 - - TTCAGGCGGC TTATCTAACA AAGCTATTAC AGAGTTAGAT AGCTTTTCGG CT -            #AGAGTTTC   1740                                                                 - - TTTGATGACG TCAACATAAT TCAACAAGTA CATGATGAAT TTTAAAGAGT TC -            #AACACTAC   1800                                                                 - - GTATGTGTTT ACTTGTTGCA GGTACGGTAA AGCTAGTTCG ATCATTTCAT GG -            #GTATCCAA   1860                                                                 - - ATAATGCTGC GGCACAACCG AAGTCGTCAA AACTTCCAAA ACAGTAGCCT TA -            #TTCCACTC   1920                                                                 - - ATTTAATTCG GGTAAAAGTT CTAGCATGTC AAAAGCGAGT TCCAAGGGAA TC -            #CTGAAGGT   1980                                                                 - - TCCATGTTAG CGTTTTTTTC GTGAATGGAA TATAAAGTAT GTAATGCAGC TA -            #CAATGACT   2040                                                                 - - TCTGGAGAGC TCGACTGTGC CTTTACAATG TCATGTAGAA TGCTTGATAA CC -            #CCAATACC   2100                                                                 - - CTTTCATGAT CAATTTCATC TAAATCCAAC AGTGCGTAAA TTGCTGTCCT CG -            #TCACTTGT   2160                                                                 - - TCAGGTGGAG ACTTGTGATT TACCAATGAA ATGATACAGT CGAAGGCCTG AT -            #CAGATAGC   2220                                                                 - - TCTTTCACCG GGACTAATAC CAGAGTTCTT AGTGCCATTA TTTGTAACTT TT -            #CATCTCTG   2280                                                                 - - CTTTTGAAAT CGTCCATTAT AAATGGCAAA GCCTCTCTGG CCTGCTGAGG TT -            #TTAATGCG   2340                                                                 - - CCGATCACCC TAATATACTC ATGGCAAATT CTTTTCACTT CTAGATCATC TT -            #CAATTTGC   2400                                                                 - - CAAAATTTCA AGAGCTCAGA AAACAGAAGG GACATTTCGC CATAGTTTCC TA -            #GAACCAAA   2460                                                                 - - TTGGCGATAA TTTTTCTCAG AGCATTTTTC CTTCTTGTTA TATTCGATTT AA -            #ACTTTTTT   2520                                                                 - - ACTCCAAAAT GTTGCAGATC TGTGACGATT TCATTTGCTT TATATCTGGC AA -            #AAACTTTT   2580                                                                 - - TGATCGGACA TAAGCGAAAT ACGTCCTATT AATGAAGTGA ATGTTCTTGC TG -            #TATTCCCT   2640                                                                 - - TCTTGTGCAG TAGATTAATT CTGTTTCCAG GCTGCGATAC TTTGATACCC AA -            #TACTAAAA   2700                                                                 - - GTTGATGATT TGAACGATCT CCTATTTCCT CGCACATTTT TGGAGCGATA CC -            #CGGAAGAC   2760                                                                 - - AGAATCGCGA TGTTAAGAAA ATAGTTCTGA TGGCACTAAA GAGATCATGA TT -            #AAGGAAAG   2820                                                                 - - GTAAGTGATA TGCATGAATG GGAATAGGCT TTCGAACTTG ACGATTTAGT TC -            #CTTATTTC   2880                                                                 - - TATCCATCTA ATCCTCCAAC TTCAATAGGC CTTATCTAGC TCAGAGCAGT AT -            #TTAATTGA   2940                                                                 - - GAATAGTAGC TTAATTGAAA CCTTACTAAA AAAGTGTATG GTTACATAAG AT -            #AAGGCGTT   3000                                                                 - - AAGAAGAGTA TACATATGCA TTATTCATTA CCAAGACCAC TATGAATAGT AA -            #TACCATAT   3060                                                                 - - TTAGCTTTTG AAACTCATGT TTTCTATTGT GTTGTTTCAA ATTCCTCTGT TA -            #GGCTCAAT   3120                                                                 - - TTAGGTTAAT TAAATTATAA AAAAATATAA AAAATAAAGA AAGTTTATCC AT -            #CGGCACCT   3180                                                                 - - CAATTCAATG GAGTAAACAG TTTCAACACT GAGTGGTGAA ACATTGAACA AC -            #TACATGCA   3240                                                                 - - GTTTCCCGCC ACGAGGCAAG TGTAGGTCCT TTGTCCATTT CGCTTTGTTT TG -            #CAGGTCAT   3300                                                                 - - TGATGACCTA ATTAGGAAGG TAGAAGCCGC TCCAGCTCAA TAAGGAAATG CT -            #AAGGGTAC   3360                                                                 - - TCGCCTTTGG TGTTTTACCA TACAATGGCA GCTTTATGTC ACTTCATTCT TC -            #AGTAACGG   3420                                                                 - - CGCTTAAATA TTCCCAAAAA CGTTACAATG GAATTGTTTG ATCATGTAAC GA -            #AATGCAAT   3480                                                                 - - CTTCTAAAAA AAAAGCCATG TGAATCAAAA AAAGATTCCT TTTAGCATAC TA -            #TAAATATG   3540                                                                 - - CAAAATGCCC TCTATTTATT CTAGTAATCG TCCATTCTCA TATCTTCCTT AT -            #ATCAGTCG   3600                                                                 - - CCTCGCTTAA TATAGTCAGC ACAAAAGGAA CAACAATTCG CCAGTTTTCA AA - # ATG           3655                                                                                        - #                  - #                  - #    Met                          - #                  - #                  - #      1         - - TTC ACT TTT CTC AAA ATT ATT CTG TGG CTT TT - #T TCC TTG GCA TTG GCC         3703                                                                       Phe Thr Phe Leu Lys Ile Ile Leu Trp Leu Ph - #e Ser Leu Ala Leu Ala                         5    - #              10    - #              15                  - - TCT GCT ATA AAT ATC AAC GAT ATC ACA TTT TC - #C AAT TTA GAA ATT ACT         3751                                                                       Ser Ala Ile Asn Ile Asn Asp Ile Thr Phe Se - #r Asn Leu Glu Ile Thr                    20         - #         25         - #         30                      - - CCA CTG ACT GCA AAT AAA CAA CCT GAT CAA GG - #T TGG ACT GCC ACT TTT         3799                                                                       Pro Leu Thr Ala Asn Lys Gln Pro Asp Gln Gl - #y Trp Thr Ala Thr Phe                35             - #     40             - #     45                          - - GAT TTT AGT ATT GCA GAT GCG TCT TCC ATT AG - #G GAG GGC GAT GAA TTC         3847                                                                       Asp Phe Ser Ile Ala Asp Ala Ser Ser Ile Ar - #g Glu Gly Asp Glu Phe            50                 - # 55                 - # 60                 - # 65       - - ACA TTA TCA ATG CCA CAT GTT TAT AGG ATT AA - #G CTA TTA AAC TCA TCG         3895                                                                       Thr Leu Ser Met Pro His Val Tyr Arg Ile Ly - #s Leu Leu Asn Ser Ser                            70 - #                 75 - #                 80              - - CAA ACA GCT ACT ATT TCC TTA GCG GAT GGT AC - #T GAG GCT TTC AAA TGC         3943                                                                       Gln Thr Ala Thr Ile Ser Leu Ala Asp Gly Th - #r Glu Ala Phe Lys Cys                        85     - #             90     - #             95                  - - TAT GTT TCG CAA CAG GCT GCA TAC TTG TAT GA - #A AAT ACT ACT TTC ACA         3991                                                                       Tyr Val Ser Gln Gln Ala Ala Tyr Leu Tyr Gl - #u Asn Thr Thr Phe Thr                   100          - #       105          - #       110                      - - TGT ACT GCT CAA AAT GAC CTG TCC TCC TAT AA - #T ACG ATT GAT GGA TCC         4039                                                                       Cys Thr Ala Gln Asn Asp Leu Ser Ser Tyr As - #n Thr Ile Asp Gly Ser               115              - #   120              - #   125                          - - ATA ACA TTT TCG CTA AAT TTT AGT GAT GGT GG - #T TCC AGC TAT GAA TAT         4087                                                                       Ile Thr Phe Ser Leu Asn Phe Ser Asp Gly Gl - #y Ser Ser Tyr Glu Tyr           130                 1 - #35                 1 - #40                 1 -      #45                                                                              - - GAG TTA GAA AAC GCT AAG TTT TTC AAA TCT GG - #G CCA ATG CTT GTT        AAA     4135                                                                    Glu Leu Glu Asn Ala Lys Phe Phe Lys Ser Gl - #y Pro Met Leu Val Lys                          150  - #               155  - #               160              - - CTT GGT AAT CAA ATG TCA GAT GTG GTG AAT TT - #C GAT CCT GCT GCT TTT         4183                                                                       Leu Gly Asn Gln Met Ser Asp Val Val Asn Ph - #e Asp Pro Ala Ala Phe                       165      - #       170          - #           175                  - - ACA GAG AAT GTT TTT CAC TCT GGG CGT TCA AC - #T GGT TAC GGT TCT TTT         4231                                                                       Thr Glu Asn Val Phe His Ser Gly Arg Ser Th - #r Gly Tyr Gly Ser Phe                   180          - #       185          - #       190                      - - GAA AGT TAT CAT TTG GGT ATG TAT TGT CCA AA - #C GGA TAT TTC CTG GGT         4279                                                                       Glu Ser Tyr His Leu Gly Met Tyr Cys Pro As - #n Gly Tyr Phe Leu Gly               195              - #   200              - #   205                          - - GGT ACT GAG AAG ATT GAT TAC GAC AGT TCC AA - #T AAC AAT GTC GAT TTG         4327                                                                       Gly Thr Glu Lys Ile Asp Tyr Asp Ser Ser As - #n Asn Asn Val Asp Leu           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - GAT TGT TCT TCA GTT CAG GTT TAT TCA TCC AA - #T GAT TTT AAT GAT        TGG     4375                                                                    Asp Cys Ser Ser Val Gln Val Tyr Ser Ser As - #n Asp Phe Asn Asp Trp                          230  - #               235  - #               240              - - TGG TTC CCG CAA AGT TAC AAT GAT ACC AAT GC - #T GAC GTC ACT TGT TTT         4423                                                                       Trp Phe Pro Gln Ser Tyr Asn Asp Thr Asn Al - #a Asp Val Thr Cys Phe                       245      - #           250      - #           255                  - - GGT AGT AAT CTG TGG ATT ACA CTT GAC GAA AA - #A CTA TAT GAT GGG GAA         4471                                                                       Gly Ser Asn Leu Trp Ile Thr Leu Asp Glu Ly - #s Leu Tyr Asp Gly Glu                   260          - #       265          - #       270                      - - ATG TTA TGG GTT AAT GCA TTA CAA TCT CTA CC - #C GCT AAT GTA AAC ACA         4519                                                                       Met Leu Trp Val Asn Ala Leu Gln Ser Leu Pr - #o Ala Asn Val Asn Thr               275              - #   280              - #   285                          - - ATA GAT CAT GCG TTA GAA TTT CAA TAC ACA TG - #C CTT GAT ACC ATA GCA         4567                                                                       Ile Asp His Ala Leu Glu Phe Gln Tyr Thr Cy - #s Leu Asp Thr Ile Ala           290                 2 - #95                 3 - #00                 3 -      #05                                                                              - - AAT ACT ACG TAC GCT ACG CAA TTC TCG ACT AC - #T AGG GAA TTT ATT        GTT     4615                                                                    Asn Thr Thr Tyr Ala Thr Gln Phe Ser Thr Th - #r Arg Glu Phe Ile Val                          310  - #               315  - #               320              - - TAT CAG GGT CGG AAC CTC GGT ACA GCT AGC GC - #C AAA AGC TCT TTT ATC         4663                                                                       Tyr Gln Gly Arg Asn Leu Gly Thr Ala Ser Al - #a Lys Ser Ser Phe Ile                       325      - #           330      - #           335                  - - TCA ACC ACT ACT ACT GAT TTA ACA AGT ATA AA - #C ACT AGT GCG TAT TCC         4711                                                                       Ser Thr Thr Thr Thr Asp Leu Thr Ser Ile As - #n Thr Ser Ala Tyr Ser                   340          - #       345          - #       350                      - - ACT GGA TCC ATT TCC ACA GTA GAA ACA GGC AA - #T CGA ACT ACA TCA GAA         4759                                                                       Thr Gly Ser Ile Ser Thr Val Glu Thr Gly As - #n Arg Thr Thr Ser Glu               355              - #   360              - #   365                          - - GTG ATC AGT CAT GTG GTG ACT ACC AGC ACA AA - #A CTG TCT CCA ACT GCT         4807                                                                       Val Ile Ser His Val Val Thr Thr Ser Thr Ly - #s Leu Ser Pro Thr Ala           370                 3 - #75                 3 - #80                 3 -      #85                                                                              - - ACT ACC AGC CTG ACA ATT GCA CAA ACC AGT AT - #C TAT TCT ACT GAC        TCA     4855                                                                    Thr Thr Ser Leu Thr Ile Ala Gln Thr Ser Il - #e Tyr Ser Thr Asp Ser                          390  - #               395  - #               400              - - AAT ATC ACA GTA GGA ACA GAT ATT CAC ACC AC - #A TCA GAA GTG ATT AGT         4903                                                                       Asn Ile Thr Val Gly Thr Asp Ile His Thr Th - #r Ser Glu Val Ile Ser                       405      - #           410      - #           415                  - - GAT GTG GAA ACC ATT AGC AGA GAA ACA GCT TC - #G ACC GTT GTA GCC GCT         4951                                                                       Asp Val Glu Thr Ile Ser Arg Glu Thr Ala Se - #r Thr Val Val Ala Ala                   420          - #       425          - #       430                      - - CCA ACC TCA ACA ACT GGA TGG ACA GGC GCT AT - #G AAT ACT TAC ATC CCG         4999                                                                       Pro Thr Ser Thr Thr Gly Trp Thr Gly Ala Me - #t Asn Thr Tyr Ile Pro               435              - #   440              - #   445                          - - CAA TTT ACA TCC TCT TCT TTC GCA ACA ATC AA - #C AGC ACA CCA ATA ATC         5047                                                                       Gln Phe Thr Ser Ser Ser Phe Ala Thr Ile As - #n Ser Thr Pro Ile Ile           450                 4 - #55                 4 - #60                 4 -      #65                                                                              - - TCT TCA TCA GCA GTA TTT GAA ACC TCA GAT GC - #T TCA ATT GTC AAT        GTG     5095                                                                    Ser Ser Ser Ala Val Phe Glu Thr Ser Asp Al - #a Ser Ile Val Asn Val                          470  - #               475  - #               480              - - CAC ACT GAA AAT ATC ACG AAT ACT GCT GCT GT - #T CCA TCT GAA GAG CCC         5143                                                                       His Thr Glu Asn Ile Thr Asn Thr Ala Ala Va - #l Pro Ser Glu Glu Pro                       485      - #           490      - #           495                  - - ACT TTT GTA AAT GCC ACG AGA AAC TCC TTA AA - #T TCC TTC TGC AGC AGC         5191                                                                       Thr Phe Val Asn Ala Thr Arg Asn Ser Leu As - #n Ser Phe Cys Ser Ser                   500          - #       505          - #       510                      - - AAA CAG CCA TCC AGT CCC TCA TCT TAT ACG TC - #T TCC CCA CTC GTA TCG         5239                                                                       Lys Gln Pro Ser Ser Pro Ser Ser Tyr Thr Se - #r Ser Pro Leu Val Ser               515              - #   520              - #   525                          - - TCC CTC TCC GTA AGC AAA ACA TTA CTA AGC AC - #C AGT TTT ACG CCT TCT         5287                                                                       Ser Leu Ser Val Ser Lys Thr Leu Leu Ser Th - #r Ser Phe Thr Pro Ser           530                 5 - #35                 5 - #40                 5 -      #45                                                                              - - GTG CCA ACA TCT AAT ACA TAT ATC AAA ACG GA - #A AAT ACG GGT TAC        TTT     5335                                                                    Val Pro Thr Ser Asn Thr Tyr Ile Lys Thr Gl - #u Asn Thr Gly Tyr Phe                          550  - #               555  - #               560              - - GAG CAC ACG GCT TTG ACA ACA TCT TCA GTT GG - #C CTT AAT TCT TTT AGT         5383                                                                       Glu His Thr Ala Leu Thr Thr Ser Ser Val Gl - #y Leu Asn Ser Phe Ser                       565      - #           570      - #           575                  - - GAA ACA GCA CTC TCA TCT CAG GGA ACG AAA AT - #T GAC ACC TTT TTA GTG         5431                                                                       Glu Thr Ala Leu Ser Ser Gln Gly Thr Lys Il - #e Asp Thr Phe Leu Val                   580          - #       585          - #       590                      - - TCA TCC TTG ATC GCA TAT CCT TCT TCT GCA TC - #A GGA AGC CAA TTG TCC         5479                                                                       Ser Ser Leu Ile Ala Tyr Pro Ser Ser Ala Se - #r Gly Ser Gln Leu Ser               595              - #   600              - #   605                          - - GGT ATC CAA CAG AAT TTC ACA TCA ACT TCT CT - #C ATG ATT TCA ACC TAT         5527                                                                       Gly Ile Gln Gln Asn Phe Thr Ser Thr Ser Le - #u Met Ile Ser Thr Tyr           610                 6 - #15                 6 - #20                 6 -      #25                                                                              - - GAA GGT AAA GCG TCT ATA TTT TTC TCA GCT GA - #G CTC GGT TCG ATC        ATT     5575                                                                    Glu Gly Lys Ala Ser Ile Phe Phe Ser Ala Gl - #u Leu Gly Ser Ile Ile                          630  - #               635  - #               640              - - TTT CTG CTT TTG TCG TAC CTG CTA TTC TAAAACGGG - #T ACTGTACAGT               5622                                                                       Phe Leu Leu Leu Ser Tyr Leu Leu Phe                                                       645      - #           650                                         - - TAGTACATTG AGTCGAAATA TACGAAATTA TTGTTCATAA TTTTCATCCT GG -             #CTCTTTTT   5682                                                                 - - TTCTTCAACC ATAGTTAAAT GGACAGTTCA TATCTTAAAC TCTAATAATA CT -            #TTTCTAGT   5742                                                                 - - TCTTATCCTT TTCCGTCTCA CCGCAGATTT TATCATAGTA TTAAATTTAT AT -            #TTTGTTCG   5802                                                                 - - TAAAAAGAAA AATTTGTGAG CGTTACCGCT CGTTTCATTA CCCGAAGGCT GT -            #TTCAGTAG   5862                                                                 - - ACCACTGATT AAGTAAGTAG ATGAAAAAAT TTCATCACCA TGAAAGAGTT CG -            #ATGAGAGC   5922                                                                 - - TACTTTTTCA AATGCTTAAC AGCTAACCGC CATTCAATAA TGTTACGTTC TC -            #TTCATTCT   5982                                                                 - - GCGGCTACGT TATCTAACAA GAGGTTTTAC TCTCTCATAT CTCATTCAAA TA -            #GAAAGAAC   6042                                                                 - - ATAATCAAAA AGCTT              - #                  - #                      - #  6057                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 650 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Phe Thr Phe Leu Lys Ile Ile Leu Trp Le - #u Phe Ser Leu Ala Leu        1               5 - #                 10 - #                 15              - - Ala Ser Ala Ile Asn Ile Asn Asp Ile Thr Ph - #e Ser Asn Leu Glu Ile                   20     - #             25     - #             30                  - - Thr Pro Leu Thr Ala Asn Lys Gln Pro Asp Gl - #n Gly Trp Thr Ala Thr               35         - #         40         - #         45                      - - Phe Asp Phe Ser Ile Ala Asp Ala Ser Ser Il - #e Arg Glu Gly Asp Glu           50             - #     55             - #     60                          - - Phe Thr Leu Ser Met Pro His Val Tyr Arg Il - #e Lys Leu Leu Asn Ser       65                 - # 70                 - # 75                 - # 80       - - Ser Gln Thr Ala Thr Ile Ser Leu Ala Asp Gl - #y Thr Glu Ala Phe Lys                       85 - #                 90 - #                 95              - - Cys Tyr Val Ser Gln Gln Ala Ala Tyr Leu Ty - #r Glu Asn Thr Thr Phe                  100      - #           105      - #           110                  - - Thr Cys Thr Ala Gln Asn Asp Leu Ser Ser Ty - #r Asn Thr Ile Asp Gly              115          - #       120          - #       125                      - - Ser Ile Thr Phe Ser Leu Asn Phe Ser Asp Gl - #y Gly Ser Ser Tyr Glu          130              - #   135              - #   140                          - - Tyr Glu Leu Glu Asn Ala Lys Phe Phe Lys Se - #r Gly Pro Met Leu Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Leu Gly Asn Gln Met Ser Asp Val Val As - #n Phe Asp Pro Ala        Ala                                                                                             165  - #               170  - #               175             - - Phe Thr Glu Asn Val Phe His Ser Gly Arg Se - #r Thr Gly Tyr Gly Ser                  180      - #           185      - #           190                  - - Phe Glu Ser Tyr His Leu Gly Met Tyr Cys Pr - #o Asn Gly Tyr Phe Leu              195          - #       200          - #       205                      - - Gly Gly Thr Glu Lys Ile Asp Tyr Asp Ser Se - #r Asn Asn Asn Val Asp          210              - #   215              - #   220                          - - Leu Asp Cys Ser Ser Val Gln Val Tyr Ser Se - #r Asn Asp Phe Asn Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Trp Trp Phe Pro Gln Ser Tyr Asn Asp Thr As - #n Ala Asp Val Thr        Cys                                                                                             245  - #               250  - #               255             - - Phe Gly Ser Asn Leu Trp Ile Thr Leu Asp Gl - #u Lys Leu Tyr Asp Gly                  260      - #           265      - #           270                  - - Glu Met Leu Trp Val Asn Ala Leu Gln Ser Le - #u Pro Ala Asn Val Asn              275          - #       280          - #       285                      - - Thr Ile Asp His Ala Leu Glu Phe Gln Tyr Th - #r Cys Leu Asp Thr Ile          290              - #   295              - #   300                          - - Ala Asn Thr Thr Tyr Ala Thr Gln Phe Ser Th - #r Thr Arg Glu Phe Ile      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Tyr Gln Gly Arg Asn Leu Gly Thr Ala Se - #r Ala Lys Ser Ser        Phe                                                                                             325  - #               330  - #               335             - - Ile Ser Thr Thr Thr Thr Asp Leu Thr Ser Il - #e Asn Thr Ser Ala Tyr                  340      - #           345      - #           350                  - - Ser Thr Gly Ser Ile Ser Thr Val Glu Thr Gl - #y Asn Arg Thr Thr Ser              355          - #       360          - #       365                      - - Glu Val Ile Ser His Val Val Thr Thr Ser Th - #r Lys Leu Ser Pro Thr          370              - #   375              - #   380                          - - Ala Thr Thr Ser Leu Thr Ile Ala Gln Thr Se - #r Ile Tyr Ser Thr Asp      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ser Asn Ile Thr Val Gly Thr Asp Ile His Th - #r Thr Ser Glu Val        Ile                                                                                             405  - #               410  - #               415             - - Ser Asp Val Glu Thr Ile Ser Arg Glu Thr Al - #a Ser Thr Val Val Ala                  420      - #           425      - #           430                  - - Ala Pro Thr Ser Thr Thr Gly Trp Thr Gly Al - #a Met Asn Thr Tyr Ile              435          - #       440          - #       445                      - - Pro Gln Phe Thr Ser Ser Ser Phe Ala Thr Il - #e Asn Ser Thr Pro Ile          450              - #   455              - #   460                          - - Ile Ser Ser Ser Ala Val Phe Glu Thr Ser As - #p Ala Ser Ile Val Asn      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Val His Thr Glu Asn Ile Thr Asn Thr Ala Al - #a Val Pro Ser Glu        Glu                                                                                             485  - #               490  - #               495             - - Pro Thr Phe Val Asn Ala Thr Arg Asn Ser Le - #u Asn Ser Phe Cys Ser                  500      - #           505      - #           510                  - - Ser Lys Gln Pro Ser Ser Pro Ser Ser Tyr Th - #r Ser Ser Pro Leu Val              515          - #       520          - #        525                     - - Ser Ser Leu Ser Val Ser Lys Thr Leu Leu Se - #r Thr Ser Phe Thr Pro          530              - #   535              - #   540                          - - Ser Val Pro Thr Ser Asn Thr Tyr Ile Lys Th - #r Glu Asn Thr Gly Tyr      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Phe Glu His Thr Ala Leu Thr Thr Ser Ser Va - #l Gly Leu Asn Ser        Phe                                                                                             565  - #               570  - #               575             - - Ser Glu Thr Ala Leu Ser Ser Gln Gly Thr Ly - #s Ile Asp Thr Phe Leu                  580      - #           585      - #           590                  - - Val Ser Ser Leu Ile Ala Tyr Pro Ser Ser Al - #a Ser Gly Ser Gln Leu              595          - #       600          - #       605                      - - Ser Gly Ile Gln Gln Asn Phe Thr Ser Thr Se - #r Leu Met Ile Ser Thr          610              - #   615              - #   620                          - - Tyr Glu Gly Lys Ala Ser Ile Phe Phe Ser Al - #a Glu Leu Gly Ser Ile      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Ile Phe Leu Leu Leu Ser Tyr Leu Leu Phe                                                  645  - #               650                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer lipol                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - -  GGGGCGGCCG AGGTCTCGCA AGATCTGGA        - #                  - #                29                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Part non-cod - #ing strand lipase                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - -  TTTGTCCAGG TCTTGCGAGA CCTCTCGACG AAT      - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Part coding - #strand lipase                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - -  TTCGGGTTAA TTGGGACATG TCTTTAGTGC GA      - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer lipo2                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - -  CCCCAAGCTT AAGGCTAGCA AGACATGTCC CAATTAACCC    - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 894 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Humicola - #lanuginosa                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 72..884                                                         (D) OTHER INFORMATION: - #/product= "lipase"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 72..881                                                         (D) OTHER INFORMATION: - #/product= "lipase"                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - GAATTCGTAG CGACGATATG AGGAGCTCCC TTGTGCTGTT CTTTGTCTCT GC -             #GTGGACGG     60                                                                 - - CCTTGGCCAC G GCC GAG GTC TCG CAA GAT CTG TTT - #AAC CAG TTC AAT        CTC     110                                                                                  Ala Glu Val - #Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu                         1   - #            5      - #            10                     - - TTT GCA CAG TAT TCT GCT GCC GCA TAC TGC GG - #A AAA AAC AAT GAT GCC          158                                                                       Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gl - #y Lys Asn Asn Asp Ala                15             - #     20             - #     25                          - - CCA GCT GGT ACA AAC ATT ACG TGC ACG GGA AA - #T GCC TGC CCC GAG GTA          206                                                                       Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly As - #n Ala Cys Pro Glu Val            30                 - # 35                 - # 40                 - # 45       - - GAG AAG GCG GAT GCA ACG TTT CTC TAC TCG TT - #T GAA GAC TCT GGA GTG          254                                                                       Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Ph - #e Glu Asp Ser Gly Val                            50 - #                 55 - #                 60              - - GGC GAT GTC ACC GGC TTC CTT GCT CTA GAC AA - #C ACG AAC AAA TTG ATC          302                                                                       Gly Asp Val Thr Gly Phe Leu Ala Leu Asp As - #n Thr Asn Lys Leu Ile                        65     - #             70     - #             75                  - - GTC CTC TCT TTC CGT GGC TCT CGT TCC ATA GA - #A AAC TGG ATC GGA AAT          350                                                                       Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Gl - #u Asn Trp Ile Gly Asn                    80         - #         85         - #         90                      - - CTT AAC TTC GAC TTG AAA GAA ATA AAT GAC AT - #T TGC TCC GGC TGC AGG          398                                                                       Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Il - #e Cys Ser Gly Cys Arg                95             - #    100             - #    105                          - - GGA CAT GAC GGC TTC ACC TCG AGC TGG AGG TC - #T GTA GCC GAT ACG TTA          446                                                                       Gly His Asp Gly Phe Thr Ser Ser Trp Arg Se - #r Val Ala Asp Thr Leu           110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - AGG CAG AAG GTG GAG GAT GCT GTG AGG GAG CA - #T CCC GAC TAT CGC        GTG      494                                                                    Arg Gln Lys Val Glu Asp Ala Val Arg Glu Hi - #s Pro Asp Tyr Arg Val                          130  - #               135  - #               140              - - GTG TTT ACC GGA CAT AGC TTG GGT GGT GCA TT - #G GCA ACT GTT GCC GGA          542                                                                       Val Phe Thr Gly His Ser Leu Gly Gly Ala Le - #u Ala Thr Val Ala Gly                       145      - #           150      - #           155                  - - GCA GAC CTG CGT GGA AAT GGG TAT GAC ATC GA - #C GTG TTT TCA TAT GGC          590                                                                       Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile As - #p Val Phe Ser Tyr Gly                   160          - #       165          - #       170                      - - GCC CCC CGA GTC GGA AAC AGG GCT TTT GCA GA - #A TTC CTG ACC GTA CAG          638                                                                       Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Gl - #u Phe Leu Thr Val Gln               175              - #   180              - #   185                          - - ACC GGC GGT ACC CTC TAC CGC ATT ACC CAC AC - #C AAT GAT ATT GTC CCT          686                                                                       Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Th - #r Asn Asp Ile Val Pro           190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - AGA CTC CCG CCG CGC GAG TTC GGT TAC AGC CA - #T TCT AGC CCA GAG        TAC      734                                                                    Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser Hi - #s Ser Ser Pro Glu Tyr                          210  - #               215  - #               220              - - TGG ATC AAA TCT GGA ACC CTT GTC CCC GTC AC - #C CGA AAC GAC ATC GTG          782                                                                       Trp Ile Lys Ser Gly Thr Leu Val Pro Val Th - #r Arg Asn Asp Ile Val                       225      - #           230      - #           235                  - - AAG ATA GAA GGC ATC GAT GCC ACC GGC GGC AA - #T AAC CAG CCT AAC ATT          830                                                                       Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly As - #n Asn Gln Pro Asn Ile                   240          - #       245          - #       250                      - - CCG GAT ATC CCT GCG CAC CTA TGG TAC TTC GG - #G TTA ATT GGG ACA TGT          878                                                                       Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gl - #y Leu Ile Gly Thr Cys               255              - #   260              - #   265                          - - CTT TAGTGCGAAG CTT            - #                  - #                      - #   894                                                                  Leu                                                                           270                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 270 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - Ala Glu Val Ser Gln Asp Leu Phe Asn Gln Ph - #e Asn Leu Phe Ala Gln        1               5 - #                 10 - #                 15              - - Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn As - #n Asp Ala Pro Ala Gly                   20     - #             25     - #             30                  - - Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pr - #o Glu Val Glu Lys Ala               35         - #         40         - #         45                      - - Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Se - #r Gly Val Gly Asp Val           50             - #     55             - #     60                          - - Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Ly - #s Leu Ile Val Leu Ser       65                 - # 70                 - # 75                 - # 80       - - Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Il - #e Gly Asn Leu Asn Phe                       85 - #                 90 - #                 95              - - Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gl - #y Cys Arg Gly His Asp                  100      - #           105      - #           110                  - - Gly Phe Thr Ser Ser Trp Arg Ser Val Ala As - #p Thr Leu Arg Gln Lys              115          - #       120          - #       125                      - - Val Glu Asp Ala Val Arg Glu His Pro Asp Ty - #r Arg Val Val Phe Thr          130              - #   135              - #   140                          - - Gly His Ser Leu Gly Gly Ala Leu Ala Thr Va - #l Ala Gly Ala Asp Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Se - #r Tyr Gly Ala Pro        Arg                                                                                             165  - #               170  - #               175             - - Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Th - #r Val Gln Thr Gly Gly                  180      - #           185      - #           190                  - - Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Il - #e Val Pro Arg Leu Pro              195          - #       200          - #       205                      - - Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pr - #o Glu Tyr Trp Ile Lys          210              - #   215              - #   220                          - - Ser Gly Thr Leu Val Pro Val Thr Arg Asn As - #p Ile Val Lys Ile Glu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pr - #o Asn Ile Pro Asp        Ile                                                                                             245  - #               250  - #               255             - - Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gl - #y Thr Cys Leu                          260      - #           265      - #           270                  - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - ATCCCTGCGC ACCTATGGTA CTTCGGGTTA ATTGGGACAT GTCTTGCTAG CC - #TTA              55                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - -  AGCTTAAGGC TAGCAAGACA TGTCCCAATT AACCCGAAGT ACCATAGGTG - #CGCAGGGAT         59                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1828 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA.                                     - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Geotrichum - #candidum                                          (B) STRAIN: CMICC 33542 - #6                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 40..1731                                                        (D) OTHER INFORMATION: - #/product= "lipase"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 40..96                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 97..1728                                                        (D) OTHER INFORMATION: - #/product= "lipase"                                       /gene= - #"lipB"                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - AATTCGGCAC GAGATTCCTT TGATTTGCAA CTGTTAATC ATG GTT TCC - # AAA AGC            54                                                                                         - #                  - #       Met Val Ser Lys Ser                            - #                  - #       -19          - #   -15        - - TTT TTT TTG GCT GCG GCG CTC AAC GTA GTG GG - #C ACC TTG GCC CAG GCC          102                                                                       Phe Phe Leu Ala Ala Ala Leu Asn Val Val Gl - #y Thr Leu Ala Gln Ala                           -10  - #                -5  - #                 1              - - CCC ACG GCC GTT CTT AAT GGC AAC GAG GTC AT - #C TCT GGT GTC CTT GAG          150                                                                       Pro Thr Ala Val Leu Asn Gly Asn Glu Val Il - #e Ser Gly Val Leu Glu                     5        - #          10        - #          15                      - - GGC AAG GTT GAT ACC TTC AAG GGA ATC CCA TT - #T GCT GAC CCT CCT GTT          198                                                                       Gly Lys Val Asp Thr Phe Lys Gly Ile Pro Ph - #e Ala Asp Pro Pro Val                20             - #     25             - #     30                          - - GGT GAC TTG CGG TTC AAG CAC CCC CAG CCT TT - #C ACT GGA TCC TAC CAG          246                                                                       Gly Asp Leu Arg Phe Lys His Pro Gln Pro Ph - #e Thr Gly Ser Tyr Gln            35                 - # 40                 - # 45                 - # 50       - - GGT CTT AAG GCC AAC GAC TTC AGC TCT GCT TG - #T ATG CAG CTT GAT CCT          294                                                                       Gly Leu Lys Ala Asn Asp Phe Ser Ser Ala Cy - #s Met Gln Leu Asp Pro                            55 - #                 60 - #                 65              - - GGC AAT GCC TTT TCT TTG CTT GAC AAA GTA GT - #G GGC TTG GGA AAG ATT          342                                                                       Gly Asn Ala Phe Ser Leu Leu Asp Lys Val Va - #l Gly Leu Gly Lys Ile                        70     - #             75     - #             80                  - - CTT CCT GAT AAC CTT AGA GGC CCT CTT TAT GA - #C ATG GCC CAG GGT AGT          390                                                                       Leu Pro Asp Asn Leu Arg Gly Pro Leu Tyr As - #p Met Ala Gln Gly Ser                    85         - #         90         - #         95                      - - GTC TCC ATG AAT GAG GAC TGT CTC TAC CTT AA - #C GTT TTC CGC CCC GCT          438                                                                       Val Ser Met Asn Glu Asp Cys Leu Tyr Leu As - #n Val Phe Arg Pro Ala               100              - #   105              - #   110                          - - GGC ACC AAG CCT GAT GCT AAG CTC CCC GTC AT - #G GTT TGG ATT TAC GGT          486                                                                       Gly Thr Lys Pro Asp Ala Lys Leu Pro Val Me - #t Val Trp Ile Tyr Gly           115                 1 - #20                 1 - #25                 1 -      #30                                                                              - - GGT GCC TTT GTG TTT GGT TCT TCT GCT TCT TA - #C CCT GGT AAC GGC        TAC      534                                                                    Gly Ala Phe Val Phe Gly Ser Ser Ala Ser Ty - #r Pro Gly Asn Gly Tyr                          135  - #               140  - #               145              - - GTC AAG GAG AGT GTG GAA ATG GGC CAG CCT GT - #T GTG TTT GTT TCC ATC          582                                                                       Val Lys Glu Ser Val Glu Met Gly Gln Pro Va - #l Val Phe Val Ser Ile                       150      - #           155      - #           160                  - - AAC TAC CGT ACC GGC CCC TAT GGA TTC TTG GG - #T GGT GAT GCC ATC ACC          630                                                                       Asn Tyr Arg Thr Gly Pro Tyr Gly Phe Leu Gl - #y Gly Asp Ala Ile Thr                   165          - #       170          - #       175                      - - GCT GAG GGC AAC ACC AAC GCT GGT CTG CAC GA - #C CAG CGC AAG GGT CTC          678                                                                       Ala Glu Gly Asn Thr Asn Ala Gly Leu His As - #p Gln Arg Lys Gly Leu               180              - #   185              - #   190                          - - GAG TGG GTT AGC GAC AAC ATT GCC AAC TTT GG - #T GGT GAT CCC GAC AAG          726                                                                       Glu Trp Val Ser Asp Asn Ile Ala Asn Phe Gl - #y Gly Asp Pro Asp Lys           195                 2 - #00                 2 - #05                 2 -      #10                                                                              - - GTC ATG ATT TTC GGT GAG TCC GCT GGT GCC AT - #G AGT GTT GCT CAC        CAG      774                                                                    Val Met Ile Phe Gly Glu Ser Ala Gly Ala Me - #t Ser Val Ala His Gln                          215  - #               220  - #               225              - - CTT GTT GCC TAC GGT GGT GAC AAC ACC TAC AA - #C GGA AAG CAG CTT TTC          822                                                                       Leu Val Ala Tyr Gly Gly Asp Asn Thr Tyr As - #n Gly Lys Gln Leu Phe                       230      - #           235      - #           240                  - - CAC TCT GCC ATT CTT CAG TCT GGC GGT CCT CT - #T CCT TAC TTT GAC TCT          870                                                                       His Ser Ala Ile Leu Gln Ser Gly Gly Pro Le - #u Pro Tyr Phe Asp Ser                   245          - #       250          - #       255                      - - ACT TCT GTT GGT CCC GAG AGT GCC TAC AGC AG - #A TTT GCT CAG TAT GCC          918                                                                       Thr Ser Val Gly Pro Glu Ser Ala Tyr Ser Ar - #g Phe Ala Gln Tyr Ala               260              - #   265              - #   270                          - - GGA TGT GAC ACC AGT GCC AGT GAT AAT GAC AC - #T CTG GCT TGT CTC CGC          966                                                                       Gly Cys Asp Thr Ser Ala Ser Asp Asn Asp Th - #r Leu Ala Cys Leu Arg           275                 2 - #80                 2 - #85                 2 -      #90                                                                              - - AGC AAG TCC AGC GAT GTC TTG CAC AGT GCG CA - #G AAC TCG TAT GAT        CTT     1014                                                                    Ser Lys Ser Ser Asp Val Leu His Ser Ala Gl - #n Asn Ser Tyr Asp Leu                          295  - #               300  - #               305              - - AAG GAC CTG TTT GGT CTG CTC CCT CAA TTC CT - #T GGA TTT GGT CCC AGA         1062                                                                       Lys Asp Leu Phe Gly Leu Leu Pro Gln Phe Le - #u Gly Phe Gly Pro Arg                       310      - #           315      - #           320                  - - CCC GAC GGC AAC ATT ATT CCC GAT GCC GCT TA - #T GAG CTC TAC CGC AGC         1110                                                                       Pro Asp Gly Asn Ile Ile Pro Asp Ala Ala Ty - #r Glu Leu Tyr Arg Ser                   325          - #       330          - #       335                      - - GGT AGA TAC GCC AAG GTT CCC TAC ATT ACT GG - #C AAC CAG GAG GAT GAG         1158                                                                       Gly Arg Tyr Ala Lys Val Pro Tyr Ile Thr Gl - #y Asn Gln Glu Asp Glu               340              - #   345              - #   350                          - - GGT ACT ATT CTT GCC CCC GTT GCT ATT AAT GC - #T ACC ACT ACT CCC CAT         1206                                                                       Gly Thr Ile Leu Ala Pro Val Ala Ile Asn Al - #a Thr Thr Thr Pro His           355                 3 - #60                 3 - #65                 3 -      #70                                                                              - - GTT AAG AAG TGG TTG AAG TAC ATT TGT AGC CA - #G GCT TCT GAC GCT        TCG     1254                                                                    Val Lys Lys Trp Leu Lys Tyr Ile Cys Ser Gl - #n Ala Ser Asp Ala Ser                          375  - #               380  - #               385              - - CTT GAT CGT GTT TTG TCG CTC TAC CCC GGC TC - #T TGG TCG GAG GGT TCA         1302                                                                       Leu Asp Arg Val Leu Ser Leu Tyr Pro Gly Se - #r Trp Ser Glu Gly Ser                       390      - #           395      - #           400                  - - CCA TTC CGC ACT GGT ATT CTT AAT GCT CTT AC - #C CCT CAG TTC AAG CGC         1350                                                                       Pro Phe Arg Thr Gly Ile Leu Asn Ala Leu Th - #r Pro Gln Phe Lys Arg                   405          - #       410          - #       415                      - - ATT GCT GCC ATT TTC ACT GAT TTG CTG TTC CA - #G TCT CCT CGT CGT GTT         1398                                                                       Ile Ala Ala Ile Phe Thr Asp Leu Leu Phe Gl - #n Ser Pro Arg Arg Val               420              - #   425              - #   430                          - - ATG CTT AAC GCT ACC AAG GAC GTC AAC CGC TG - #G ACT TAC CTT GCC ACC         1446                                                                       Met Leu Asn Ala Thr Lys Asp Val Asn Arg Tr - #p Thr Tyr Leu Ala Thr           435                 4 - #40                 4 - #45                 4 -      #50                                                                              - - CAG CTC CAT AAC CTC GTT CCA TTT TTG GGT AC - #T TTC CAT GGC AGT        GAT     1494                                                                    Gln Leu His Asn Leu Val Pro Phe Leu Gly Th - #r Phe His Gly Ser Asp                          455  - #               460  - #               465              - - CTT CTT TTT CAA TAC TAC GTG GAC CTT GGC CC - #A TCT TCT GCT TAC CGC         1542                                                                       Leu Leu Phe Gln Tyr Tyr Val Asp Leu Gly Pr - #o Ser Ser Ala Tyr Arg                       470      - #           475      - #           480                  - - CGC TAC TTT ATC TCG TTT GCC AAC CAC CAC GA - #C CCC AAC GTT GGT ACC         1590                                                                       Arg Tyr Phe Ile Ser Phe Ala Asn His His As - #p Pro Asn Val Gly Thr                   485          - #       490          - #       495                      - - AAC CTC CAA CAG TGG GAT ATG TAC ACT GAT GC - #A GGC AAG GAG ATG CTT         1638                                                                       Asn Leu Gln Gln Trp Asp Met Tyr Thr Asp Al - #a Gly Lys Glu Met Leu               500              - #   505              - #   510                          - - CAG ATT CAT ATG ATT GGT AAC TCT ATG AGA AC - #T GAC GAC TTT AGA ATC         1686                                                                       Gln Ile His Met Ile Gly Asn Ser Met Arg Th - #r Asp Asp Phe Arg Ile           515                 5 - #20                 5 - #25                 5 -      #30                                                                              - - GAG GGA ATC TCG AAC TTT GAG TCT GAC GTT AC - #T CTC TTC GGT            TAATCCCATT  1738                                                                Glu Gly Ile Ser Asn Phe Glu Ser Asp Val Th - #r Leu Phe Gly                                   535  - #               540  - #                545            - - TAGCAAGTTT TGTGTATTTC AAGTATACCA GTTGATGTAA TATATCAATA GA -             #TTACAAAT   1798                                                                 - - TAATTAGTGA AAAAAAAAAA AAAAAAAAAC         - #                  - #             1828                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 563 amino - #acids                                                (B) TYPE: amino acio                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - Met Val Ser Lys Ser Phe Phe Leu Ala Ala Al - #a Leu Asn Val Val Gly     19             -15    - #             -10    - #              -5                - - Thr Leu Ala Gln Ala Pro Thr Ala Val Leu As - #n Gly Asn Glu Val Ile                    1    - #           5       - #           10                      - - Ser Gly Val Leu Glu Gly Lys Val Asp Thr Ph - #e Lys Gly Ile Pro Phe           15             - #     20             - #     25                          - - Ala Asp Pro Pro Val Gly Asp Leu Arg Phe Ly - #s His Pro Gln Pro Phe       30                 - # 35                 - # 40                 - # 45       - - Thr Gly Ser Tyr Gln Gly Leu Lys Ala Asn As - #p Phe Ser Ser Ala Cys                       50 - #                 55 - #                 60              - - Met Gln Leu Asp Pro Gly Asn Ala Phe Ser Le - #u Leu Asp Lys Val Val                   65     - #             70     - #             75                  - - Gly Leu Gly Lys Ile Leu Pro Asp Asn Leu Ar - #g Gly Pro Leu Tyr Asp               80         - #         85         - #         90                      - - Met Ala Gln Gly Ser Val Ser Met Asn Glu As - #p Cys Leu Tyr Leu Asn           95             - #    100             - #    105                          - - Val Phe Arg Pro Ala Gly Thr Lys Pro Asp Al - #a Lys Leu Pro Val Met      110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - Val Trp Ile Tyr Gly Gly Ala Phe Val Phe Gl - #y Ser Ser Ala Ser        Tyr                                                                                             130  - #               135  - #               140             - - Pro Gly Asn Gly Tyr Val Lys Glu Ser Val Gl - #u Met Gly Gln Pro Val                  145      - #           150      - #           155                  - - Val Phe Val Ser Ile Asn Tyr Arg Thr Gly Pr - #o Tyr Gly Phe Leu Gly              160          - #       165          - #       170                      - - Gly Asp Ala Ile Thr Ala Glu Gly Asn Thr As - #n Ala Gly Leu His Asp          175              - #   180              - #   185                          - - Gln Arg Lys Gly Leu Glu Trp Val Ser Asp As - #n Ile Ala Asn Phe Gly      190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - Gly Asp Pro Asp Lys Val Met Ile Phe Gly Gl - #u Ser Ala Gly Ala        Met                                                                                             210  - #               215  - #               220             - - Ser Val Ala His Gln Leu Val Ala Tyr Gly Gl - #y Asp Asn Thr Tyr Asn                  225      - #           230      - #           235                  - - Gly Lys Gln Leu Phe His Ser Ala Ile Leu Gl - #n Ser Gly Gly Pro Leu              240          - #       245          - #       250                      - - Pro Tyr Phe Asp Ser Thr Ser Val Gly Pro Gl - #u Ser Ala Tyr Ser Arg          255              - #   260              - #   265                          - - Phe Ala Gln Tyr Ala Gly Cys Asp Thr Ser Al - #a Ser Asp Asn Asp Thr      270                 2 - #75                 2 - #80                 2 -      #85                                                                              - - Leu Ala Cys Leu Arg Ser Lys Ser Ser Asp Va - #l Leu His Ser Ala        Gln                                                                                             290  - #               295  - #               300             - - Asn Ser Tyr Asp Leu Lys Asp Leu Phe Gly Le - #u Leu Pro Gln Phe Leu                  305      - #           310      - #           315                  - - Gly Phe Gly Pro Arg Pro Asp Gly Asn Ile Il - #e Pro Asp Ala Ala Tyr              320          - #       325          - #       330                      - - Glu Leu Tyr Arg Ser Gly Arg Tyr Ala Lys Va - #l Pro Tyr Ile Thr Gly          335              - #   340              - #   345                          - - Asn Gln Glu Asp Glu Gly Thr Ile Leu Ala Pr - #o Val Ala Ile Asn Ala      350                 3 - #55                 3 - #60                 3 -      #65                                                                              - - Thr Thr Thr Pro His Val Lys Lys Trp Leu Ly - #s Tyr Ile Cys Ser        Gln                                                                                             370  - #               375  - #               380             - - Ala Ser Asp Ala Ser Leu Asp Arg Val Leu Se - #r Leu Tyr Pro Gly Ser                  385      - #           390      - #           395                  - - Trp Ser Glu Gly Ser Pro Phe Arg Thr Gly Il - #e Leu Asn Ala Leu Thr              400          - #       405          - #       410                      - - Pro Gln Phe Lys Arg Ile Ala Ala Ile Phe Th - #r Asp Leu Leu Phe Gln          415              - #   420              - #   425                          - - Ser Pro Arg Arg Val Met Leu Asn Ala Thr Ly - #s Asp Val Asn Arg Trp      430                 4 - #35                 4 - #40                 4 -      #45                                                                              - - Thr Tyr Leu Ala Thr Gln Leu His Asn Leu Va - #l Pro Phe Leu Gly        Thr                                                                                             450  - #               455  - #               460             - - Phe His Gly Ser Asp Leu Leu Phe Gln Tyr Ty - #r Val Asp Leu Gly Pro                  465      - #           470      - #           475                  - - Ser Ser Ala Tyr Arg Arg Tyr Phe Ile Ser Ph - #e Ala Asn His His Asp              480          - #       485          - #       490                      - - Pro Asn Val Gly Thr Asn Leu Gln Gln Trp As - #p Met Tyr Thr Asp Ala          495              - #   500              - #   505                          - - Gly Lys Glu Met Leu Gln Ile His Met Ile Gl - #y Asn Ser Met Arg Thr      510                 5 - #15                 5 - #20                 5 -      #25                                                                              - - Asp Asp Phe Arg Ile Glu Gly Ile Ser Asn Ph - #e Glu Ser Asp Val        Thr                                                                                             530  - #               535  - #               540             - - Leu Phe Gly                                                               - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer lipo3                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - GGGGCGGCCG CGCAGGCCCC AAGGCGGTCT CTCAAT      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Part non-cod - #ing strand lipaseII                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - ATTGAGAGAC CGCCGTGGGG CCTGGGCCAG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Part coding - #strand lipaseII                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                          - - CAAACTTTGA GACTGACGTT AATCTCTACG GTTAAAAC      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer lipo4                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                          - - CCCCGCTAGC ACCGTAGAGA TTAACGTCAG TC       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer lipo5                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - CCCGCGGCCG CGAGCATTGA TGGTGGTATC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Part non-cod - #ing strand lipase                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                          - - GATACCACGA TCAATGCT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 19:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nueleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Part coding - #strand lipase                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                          - - AACACAGGCC TCTGTACT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 20:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer lipo6                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                          - - CCGCGCTAGC AGTACAGAGG CCTGTGTT         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 21:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2685 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) ORIGINAL SOURCE:                                                          (A) ORGANISM: Saccharomyce - #s cerevisiae                           - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: pYY105                                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..2685                                                         (D) OTHER INFORMATION: - #/product= "Flocculation protein"         /gene=                                                                                        nFLO1"                                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                          - - ATG ACA ATG CCT CAT CGC TAT ATG TTT TTG GC - #A GTC TTT ACA CTT        CTG       48                                                                    Met Thr Met Pro His Arg Tyr Met Phe Leu Al - #a Val Phe Thr Leu Leu            1               5 - #                 10 - #                 15              - - GCA CTA ACT AGT GTG GCC TCA GGA GCC ACA GA - #G GCG TGC TTA CCA GCA           96                                                                       Ala Leu Thr Ser Val Ala Ser Gly Ala Thr Gl - #u Ala Cys Leu Pro Ala                        20     - #             25     - #             30                  - - GGC CAG AGG AAA AGT GGG ATG AAT ATA AAT TT - #T TAC CAG TAT TCA TTG          144                                                                       Gly Gln Arg Lys Ser Gly Met Asn Ile Asn Ph - #e Tyr Gln Tyr Ser Leu                    35         - #         40         - #         45                      - - AAA GAT TCC TCC ACA TAT TCG AAT GCA GCA TA - #T ATG GCT TAT GGA TAT          192                                                                       Lys Asp Ser Ser Thr Tyr Ser Asn Ala Ala Ty - #r Met Ala Tyr Gly Tyr                50             - #     55             - #     60                          - - GCC TCA AAA ACC AAA CTA GGT TCT GTC GGA GG - #A CAA ACT GAT ATC TCG          240                                                                       Ala Ser Lys Thr Lys Leu Gly Ser Val Gly Gl - #y Gln Thr Asp Ile Ser            65                 - # 70                 - # 75                 - # 80       - - ATT GAT TAT AAT ATT CCC TGT GTT AGT TCA TC - #A GGC ACA TTT CCT TGT          288                                                                       Ile Asp Tyr Asn Ile Pro Cys Val Ser Ser Se - #r Gly Thr Phe Pro Cys                            85 - #                 90 - #                 95              - - CCT CAA GAA GAT TCC TAT GGA AAC TGG GGA TG - #C AAA GGA ATG GGT GCT          336                                                                       Pro Gln Glu Asp Ser Tyr Gly Asn Trp Gly Cy - #s Lys Gly Met Gly Ala                       100      - #           105      - #           110                  - - TGT TCT AAT AGT CAA GGA ATT GCA TAC TGG AG - #T ACT GAT TTA TTT GGT          384                                                                       Cys Ser Asn Ser Gln Gly Ile Ala Tyr Trp Se - #r Thr Asp Leu Phe Gly                   115          - #       120          - #       125                      - - TTC TAT ACT ACC CCA ACA AAC GTA ACC CTA GA - #A ATG ACA GGT TAT TTT          432                                                                       Phe Tyr Thr Thr Pro Thr Asn Val Thr Leu Gl - #u Met Thr Gly Tyr Phe               130              - #   135              - #   140                          - - TTA CCA CCA CAG ACG GGT TCT TAC ACA TTC AA - #G TTT GCT ACA GTT GAC          480                                                                       Leu Pro Pro Gln Thr Gly Ser Tyr Thr Phe Ly - #s Phe Ala Thr Val Asp           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GAC TCT GCA ATT CTA TCA GTA GGT GGT GCA AC - #C GCG TTC AAC TGT        TGT      528                                                                    Asp Ser Ala Ile Leu Ser Val Gly Gly Ala Th - #r Ala Phe Asn Cys Cys                          165  - #               170  - #               175              - - GCT CAA CAG CAA CCG CCG ATC ACA TCA ACG AA - #C TTT ACC ATT GAC GGT          576                                                                       Ala Gln Gln Gln Pro Pro Ile Thr Ser Thr As - #n Phe Thr Ile Asp Gly                       180      - #           185      - #           190                  - - ATC AAG CCA TGG GGT GGA AGT TTG CCA CCT AA - #T ATC GAA GGA ACC GTC          624                                                                       Ile Lys Pro Trp Gly Gly Ser Leu Pro Pro As - #n Ile Glu Gly Thr Val                   195          - #       200          - #       205                      - - TAT ATG TAC GCT GGC TAC TAT TAT CCA ATG AA - #G GTT GTT TAC TCG AAC          672                                                                       Tyr Met Tyr Ala Gly Tyr Tyr Tyr Pro Met Ly - #s Val Val Tyr Ser Asn               210              - #   215              - #   220                          - - GCT GTT TCT TGG GGT ACA CTT CCA ATT AGT GT - #G ACA CTT CCA GAT GGT          720                                                                       Ala Val Ser Trp Gly Thr Leu Pro Ile Ser Va - #l Thr Leu Pro Asp Gly           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - ACC ACT GTA AGT GAT GAC TTC GAA GGG TAC GT - #C TAT TCC TTT GAC        GAT      768                                                                    Thr Thr Val Ser Asp Asp Phe Glu Gly Tyr Va - #l Tyr Ser Phe Asp Asp                          245  - #               250  - #               255              - - GAC CTA AGT CAA TCT AAC TGT ACT GTC CCT GA - #C CCT TCA AAT TAT GCT          816                                                                       Asp Leu Ser Gln Ser Asn Cys Thr Val Pro As - #p Pro Ser Asn Tyr Ala                       260      - #           265      - #           270                  - - GTC AGT ACC ACT ACA ACT ACA ACG GAA CCA TG - #G ACC GGT ACT TTC ACT          864                                                                       Val Ser Thr Thr Thr Thr Thr Thr Glu Pro Tr - #p Thr Gly Thr Phe Thr                   275          - #       280          - #       285                      - - TCT ACA TCT ACT GAA ATG ACC ACC GTC ACC GG - #T ACC AAC GGC GTT CCA          912                                                                       Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gl - #y Thr Asn Gly Val Pro               290              - #   295              - #   300                          - - ACT GAC GAA ACC GTC ATT GTC ATC AGA ACT CC - #A ACC AGT GAA GGT CTA          960                                                                       Thr Asp Glu Thr Val Ile Val Ile Arg Thr Pr - #o Thr Ser Glu Gly Leu           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ATC AGC ACC ACC ACT GAA CCA TGG ACT GGC AC - #T TTC ACT TCG ACT        TCC     1008                                                                    Ile Ser Thr Thr Thr Glu Pro Trp Thr Gly Th - #r Phe Thr Ser Thr Ser                          325  - #               330  - #               335              - - ACT GAG GTT ACC ACC ATC ACT GGA ACC AAC GG - #T CAA CCA ACT GAC GAA         1056                                                                       Thr Glu Val Thr Thr Ile Thr Gly Thr Asn Gl - #y Gln Pro Thr Asp Glu                       340      - #           345      - #           350                  - - ACT GTG ATT GTT ATC AGA ACT CCA ACC AGT GA - #A GGT CTA ATC AGC ACC         1104                                                                       Thr Val Ile Val Ile Arg Thr Pro Thr Ser Gl - #u Gly Leu Ile Ser Thr                   355          - #       360          - #       365                      - - ACC ACT GAA CCA TGG ACT GGT ACT TTC ACT TC - #T ACA TCT ACT GAA ATG         1152                                                                       Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Se - #r Thr Ser Thr Glu Met               370              - #   375              - #   380                          - - ACC ACC GTC ACC GGT ACT AAC GGT CAA CCA AC - #T GAC GAA ACC GTG ATT         1200                                                                       Thr Thr Val Thr Gly Thr Asn Gly Gln Pro Th - #r Asp Glu Thr Val Ile           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - GTT ATC AGA ACT CCA ACC AGT GAA GGT TTG GT - #T ACA ACC ACC ACT        GAA     1248                                                                    Val Ile Arg Thr Pro Thr Ser Glu Gly Leu Va - #l Thr Thr Thr Thr Glu                          405  - #               410  - #               415              - - CCA TGG ACT GGT ACT TTT ACT TCG ACT TCC AC - #T GAA ATG TCT ACT GTC         1296                                                                       Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Th - #r Glu Met Ser Thr Val                       420      - #           425      - #           430                  - - ACT GGA ACC AAT GGC TTG CCA ACT GAT GAA AC - #T GTC ATT GTT GTC AAA         1344                                                                       Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Th - #r Val Ile Val Val Lys                   435          - #       440          - #       445                      - - ACT CCA ACT ACT GCC ATC TCA TCC AGT TTG TC - #A TCA TCA TCT TCA GGA         1392                                                                       Thr Pro Thr Thr Ala Ile Ser Ser Ser Leu Se - #r Ser Ser Ser Ser Gly               450              - #   455              - #   460                          - - CAA ATC ACC AGC TCT ATC ACG TCT TCG CGT CC - #A ATT ATT ACC CCA TTC         1440                                                                       Gln Ile Thr Ser Ser Ile Thr Ser Ser Arg Pr - #o Ile Ile Thr Pro Phe           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - TAT CCT AGC AAT GGA ACT TCT GTG ATT TCT TC - #C TCA GTA ATT TCT        TCC     1488                                                                    Tyr Pro Ser Asn Gly Thr Ser Val Ile Ser Se - #r Ser Val Ile Ser Ser                          485  - #               490  - #               495              - - TCA GTC ACT TCT TCT CTA TTC ACT TCT TCT CC - #A GTC ATT TCT TCC TCA         1536                                                                       Ser Val Thr Ser Ser Leu Phe Thr Ser Ser Pr - #o Val Ile Ser Ser Ser                       500      - #           505      - #           510                  - - GTC ATT TCT TCT TCT ACA ACA ACC TCC ACT TC - #T ATA TTT TCT GAA TCA         1584                                                                       Val Ile Ser Ser Ser Thr Thr Thr Ser Thr Se - #r Ile Phe Ser Glu Ser                   515          - #       520          - #       525                      - - TCT AAA TCA TCC GTC ATT CCA ACC AGT AGT TC - #C ACC TCT GGT TCT TCT         1632                                                                       Ser Lys Ser Ser Val Ile Pro Thr Ser Ser Se - #r Thr Ser Gly Ser Ser               530              - #   535              - #   540                          - - GAG AGC GAA ACG AGT TCA GCT GGT TCT GTC TC - #T TCT TCC TCT TTT ATC         1680                                                                       Glu Ser Glu Thr Ser Ser Ala Gly Ser Val Se - #r Ser Ser Ser Phe Ile           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - TCT TCT GAA TCA TCA AAA TCT CCT ACA TAT TC - #T TCT TCA TCA TTA        CCA     1728                                                                    Ser Ser Glu Ser Ser Lys Ser Pro Thr Tyr Se - #r Ser Ser Ser Leu Pro                          565  - #               570  - #               575              - - CTT GTT ACC AGT GCG ACA ACA AGC CAG GAA AC - #T GCT TCT TCA TTA CCA         1776                                                                       Leu Val Thr Ser Ala Thr Thr Ser Gln Glu Th - #r Ala Ser Ser Leu Pro                       580      - #           585      - #           590                  - - CCT GCT ACC ACT ACA AAA ACG AGC GAA CAA AC - #C ACT TTG GTT ACC GTG         1824                                                                       Pro Ala Thr Thr Thr Lys Thr Ser Glu Gln Th - #r Thr Leu Val Thr Val                   595          - #       600          - #       605                      - - ACA TCC TGC GAG TCT CAT GTG TGC ACT GAA TC - #C ATC TCC CCT GCG ATT         1872                                                                       Thr Ser Cys Glu Ser His Val Cys Thr Glu Se - #r Ile Ser Pro Ala Ile               610              - #   615              - #   620                          - - GTT TCC ACA GCT ACT GTT ACT GTT AGC GGC GT - #C ACA ACA GAG TAT ACC         1920                                                                       Val Ser Thr Ala Thr Val Thr Val Ser Gly Va - #l Thr Thr Glu Tyr Thr           625                 6 - #30                 6 - #35                 5 -      #40                                                                              - - ACA TGG TGC CCT ATT TCT ACT ACA GAG ACA AC - #A AAG CAA ACC AAA        GGG     1968                                                                    Thr Trp Cys Pro Ile Ser Thr Thr Glu Thr Th - #r Lys Gln Thr Lys Gly                          645  - #               650  - #               655              - - ACA ACA GAG CAA ACC ACA GAA ACA ACA AAA CA - #A ACC ACG GTA GTT ACA         2016                                                                       Thr Thr Glu Gln Thr Thr Glu Thr Thr Lys Gl - #n Thr Thr Val Val Thr                       660      - #           665      - #           670                  - - ATT TCT TCT TGT GAA TCT GAC GTA TGC TCT AA - #G ACT GCT TCT CCA GCC         2064                                                                       Ile Ser Ser Cys Glu Ser Asp Val Cys Ser Ly - #s Thr Ala Ser Pro Ala                   675          - #       680          - #       685                      - - ATT GTA TCT ACA AGC ACT GCT ACT ATT AAC GG - #C GTT ACT ACA GAA TAC         2112                                                                       Ile Val Ser Thr Ser Thr Ala Thr Ile Asn Gl - #y Val Thr Thr Glu Tyr               690              - #   695              - #   700                          - - ACA ACA TGG TGT CCT ATT TCC ACC ACA GAA TC - #G AGG CAA CAA ACA ACG         2160                                                                       Thr Thr Trp Cys Pro Ile Ser Thr Thr Glu Se - #r Arg Gln Gln Thr Thr           705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - CTA GTT ACT GTT ACT TCC TGC GAA TCT GGT GT - #G TGT TCC GAA ACT        GCT     2208                                                                    Leu Val Thr Val Thr Ser Cys Glu Ser Gly Va - #l Cys Ser Glu Thr Ala                          725  - #               730  - #               735              - - TCA CCT GCC ATT GTT TCG ACG GCC ACG GCT AC - #T GTG AAT GAT GTT GTT         2256                                                                       Ser Pro Ala Ile Val Ser Thr Ala Thr Ala Th - #r Val Asn Asp Val Val                       740      - #           745      - #           750                  - - ACG GTC TAT CCT ACA TGG AGG CCA CAG ACT GC - #G AAT GAA GAG TCT GTC         2304                                                                       Thr Val Tyr Pro Thr Trp Arg Pro Gln Thr Al - #a Asn Glu Glu Ser Val                   755          - #       760          - #       765                      - - AGC TCT AAA ATG AAC AGT GCT ACC GGT GAG AC - #A ACA ACC AAT ACT TTA         2352                                                                       Ser Ser Lys Met Asn Ser Ala Thr Gly Glu Th - #r Thr Thr Asn Thr Leu               770              - #   775              - #   780                          - - GCT GCT GAA ACG ACT ACC AAT ACT GTA GCT GC - #T GAG ACG ATT ACC AAT         2400                                                                       Ala Ala Glu Thr Thr Thr Asn Thr Val Ala Al - #a Glu Thr Ile Thr Asn           785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - ACT GGA GCT GCT GAG ACG AAA ACA GTA GTC AC - #C TCT TCG CTT TCA        AGA     2448                                                                    Thr Gly Ala Ala Glu Thr Lys Thr Val Val Th - #r Ser Ser Leu Ser Arg                          805  - #               810  - #               815              - - TCT AAT CAC GCT GAA ACA CAG ACG GCT TCC GC - #G ACC GAT GTG ATT GGT         2496                                                                       Ser Asn His Ala Glu Thr Gln Thr Ala Ser Al - #a Thr Asp Val Ile Gly                       820      - #           825      - #           830                  - - CAC AGC AGT AGT GTT GTT TCT GTA TCC GAA AC - #T GGC AAC ACC AAG AGT         2544                                                                       His Ser Ser Ser Val Val Ser Val Ser Glu Th - #r Gly Asn Thr Lys Ser                   835          - #       840          - #       845                      - - CTA ACA AGT TCC GGG TTG AGT ACT ATG TCG CA - #A CAG CCT CGT AGC ACA         2592                                                                       Leu Thr Ser Ser Gly Leu Ser Thr Met Ser Gl - #n Gln Pro Arg Ser Thr               850              - #   855              - #   860                          - - CCA GCA AGC AGC ATG GTA GGA TAT AGT ACA GC - #T TCT TTA GAA ATT TCA         2640                                                                       Pro Ala Ser Ser Met Val Gly Tyr Ser Thr Al - #a Ser Leu Glu Ile Ser           865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - ACG TAT GCT GGC AGT GCA ACA GCT TAC TGG CC - #G GTA GTG GTT TAA             2685                                                                      Thr Tyr Ala Gly Ser Ala Thr Ala Tyr Trp Pr - #o Val Val Val                                   885  - #               890                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 22:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 894 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                          - - Met Thr Met Pro His Arg Tyr Met Phe Leu Al - #a Val Phe Thr Leu Leu        1               5 - #                 10 - #                 15              - - Ala Leu Thr Ser Val Ala Ser Gly Ala Thr Gl - #u Ala Cys Leu Pro Ala                   20     - #             25     - #             30                  - - Gly Gln Arg Lys Ser Gly Met Asn Ile Asn Ph - #e Tyr Gln Tyr Ser Leu               35         - #         40         - #         45                      - - Lys Asp Ser Ser Thr Tyr Ser Asn Ala Ala Ty - #r Met Ala Tyr Gly Tyr           50             - #     55             - #     60                          - - Ala Ser Lys Thr Lys Leu Gly Ser Val Gly Gl - #y Gln Thr Asp Ile Ser       65                 - # 70                 - # 75                 - # 80       - - Ile Asp Tyr Asn Ile Pro Cys Val Ser Ser Se - #r Gly Thr Phe Pro Cys                       85 - #                 90 - #                 95              - - Pro Gln Glu Asp Ser Tyr Gly Asn Trp Gly Cy - #s Lys Gly Met Gly Ala                  100      - #           105      - #           110                  - - Cys Ser Asn Ser Gln Gly Ile Ala Tyr Trp Se - #r Thr Asp Leu Phe Gly              115          - #       120          - #       125                      - - Phe Tyr Thr Thr Pro Thr Asn Val Thr Leu Gl - #u Met Thr Gly Tyr Phe          130              - #   135              - #   140                          - - Leu Pro Pro Gln Thr Gly Ser Tyr Thr Phe Ly - #s Phe Ala Thr Val Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Ser Ala Ile Leu Ser Val Gly Gly Ala Th - #r Ala Phe Asn Cys        Cys                                                                                             165  - #               170  - #               175             - - Ala Gln Gln Gln Pro Pro Ile Thr Ser Thr As - #n Phe Thr Ile Asp Gly                  180      - #           185      - #           190                  - - Ile Lys Pro Trp Gly Gly Ser Leu Pro Pro As - #n Ile Glu Gly Thr Val              195          - #       200          - #       205                      - - Tyr Met Tyr Ala Gly Tyr Tyr Tyr Pro Met Ly - #s Val Val Tyr Ser Asn          210              - #   215              - #   220                          - - Ala Val Ser Trp Gly Thr Leu Pro Ile Ser Va - #l Thr Leu Pro Asp Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Thr Thr Val Ser Asp Asp Phe Glu Gly Tyr Va - #l Tyr Ser Phe Asp        Asp                                                                                             245  - #               250  - #               255             - - Asp Leu Ser Gln Ser Asn Cys Thr Val Pro As - #p Pro Ser Asn Tyr Ala                  260      - #           265      - #           270                  - - Val Ser Thr Thr Thr Thr Thr Thr Glu Pro Tr - #p Thr Gly Thr Phe Thr              275          - #       280          - #       285                      - - Ser Thr Ser Thr Glu Met Thr Thr Val Thr Gl - #y Thr Asn Gly Val Pro          290              - #   295              - #   300                          - - Thr Asp Glu Thr Val Ile Val Ile Arg Thr Pr - #o Thr Ser Glu Gly Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ile Ser Thr Thr Thr Glu Pro Trp Thr Gly Th - #r Phe Thr Ser Thr        Ser                                                                                             325  - #               330  - #               335             - - Thr Glu Val Thr Thr Ile Thr Gly Thr Asn Gl - #y Gln Pro Thr Asp Glu                  340      - #           345      - #           350                  - - Thr Val Ile Val Ile Arg Thr Pro Thr Ser Gl - #u Gly Leu Ile Ser Thr              355          - #       360          - #       365                      - - Thr Thr Glu Pro Trp Thr Gly Thr Phe Thr Se - #r Thr Ser Thr Glu Met          370              - #   375              - #   380                          - - Thr Thr Val Thr Gly Thr Asn Gly Gln Pro Th - #r Asp Glu Thr Val Ile      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Val Ile Arg Thr Pro Thr Ser Glu Gly Leu Va - #l Thr Thr Thr Thr        Glu                                                                                             405  - #               410  - #               415             - - Pro Trp Thr Gly Thr Phe Thr Ser Thr Ser Th - #r Glu Met Ser Thr Val                  420      - #           425      - #           430                  - - Thr Gly Thr Asn Gly Leu Pro Thr Asp Glu Th - #r Val Ile Val Val Lys              435          - #       440          - #       445                      - - Thr Pro Thr Thr Ala Ile Ser Ser Ser Leu Se - #r Ser Ser Ser Ser Gly          450              - #   455              - #   460                          - - Gln Ile Thr Ser Ser Ile Thr Ser Ser Arg Pr - #o Ile Ile Thr Pro Phe      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Tyr Pro Ser Asn Gly Thr Ser Val Ile Ser Se - #r Ser Val Ile Ser        Ser                                                                                             485  - #               490  - #               495             - - Ser Val Thr Ser Ser Leu Phe Thr Ser Ser Pr - #o Val Ile Ser Ser Ser                  500      - #           505      - #           510                  - - Val Ile Ser Ser Ser Thr Thr Thr Ser Thr Se - #r Ile Phe Ser Glu Ser              515          - #       520          - #       525                      - - Ser Lys Ser Ser Val Ile Pro Thr Ser Ser Se - #r Thr Ser Gly Ser Ser          530              - #   535              - #   540                          - - Glu Ser Glu Thr Ser Ser Ala Gly Ser Val Se - #r Ser Ser Ser Phe Ile      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Ser Ser Glu Ser Ser Lys Ser Pro Thr Tyr Se - #r Ser Ser Ser Leu        Pro                                                                                             565  - #               570  - #               575             - - Leu Val Thr Ser Ala Thr Thr Ser Gln Glu Th - #r Ala Ser Ser Leu Pro                  580      - #           585      - #           590                  - - Pro Ala Thr Thr Thr Lys Thr Ser Glu Gln Th - #r Thr Leu Val Thr Val              595          - #       600          - #       605                      - - Thr Ser Cys Glu Ser His Val Cys Thr Glu Se - #r Ile Ser Pro Ala Ile          610              - #   615              - #   620                          - - Val Ser Thr Ala Thr Val Thr Val Ser Gly Va - #l Thr Thr Glu Tyr Thr      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Thr Trp Cys Pro Ile Ser Thr Thr Glu Thr Th - #r Lys Gln Thr Lys        Gly                                                                                             645  - #               650  - #               655             - - Thr Thr Glu Gln Thr Thr Glu Thr Thr Lys Gl - #n Thr Thr Val Val Thr                  660      - #           665      - #           670                  - - Ile Ser Ser Cys Glu Ser Asp Val Cys Ser Ly - #s Thr Ala Ser Pro Ala              675          - #       680          - #       685                      - - Ile Val Ser Thr Ser Thr Ala Thr Ile Asn Gl - #y Val Thr Thr Glu Tyr          690              - #   695              - #   700                          - - Thr Thr Trp Cys Pro Ile Ser Thr Thr Glu Se - #r Arg Gln Gln Thr Thr      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Leu Val Thr Val Thr Ser Cys Glu Ser Gly Va - #l Cys Ser Glu Thr        Ala                                                                                             725  - #               730  - #               735             - - Ser Pro Ala Ile Val Ser Thr Ala Thr Ala Th - #r Val Asn Asp Val Val                  740      - #           745      - #           750                  - - Thr Val Tyr Pro Thr Trp Arg Pro Gln Thr Al - #a Asn Glu Glu Ser Val              755          - #       760          - #       765                      - - Ser Ser Lys Met Asn Ser Ala Thr Gly Glu Th - #r Thr Thr Asn Thr Leu          770              - #   775              - #   780                          - - Ala Ala Glu Thr Thr Thr Asn Thr Val Ala Al - #a Glu Thr Ile Thr Asn      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Thr Gly Ala Ala Glu Thr Lys Thr Val Val Th - #r Ser Ser Leu Ser        Arg                                                                                             805  - #               810  - #               815             - - Ser Asn His Ala Glu Thr Gln Thr Ala Ser Al - #a Thr Asp Val Ile Gly                  820      - #           825      - #           830                  - - His Ser Ser Ser Val Val Ser Val Ser Glu Th - #r Gly Asn Thr Lys Ser              835          - #       840          - #       845                      - - Leu Thr Ser Ser Gly Leu Ser Thr Met Ser Gl - #n Gln Pro Arg Ser Thr          850              - #   855              - #   860                          - - Pro Ala Ser Ser Met Val Gly Tyr Ser Thr Al - #a Ser Leu Glu Ile Ser      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Thr Tyr Ala Gly Ser Ala Thr Ala Tyr Trp Pr - #o Val Val Val                             885  - #               890                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer pcrfl - #ol                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                          - - GAATTCGCTA GCAATTATGC TGTCAGTACC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 24:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Part non-cod - #ing sequence FLO1                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                          - - AGTGGTACTG ACAGCATAAT TTGA          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 25:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Part coding - #sequence FLO1                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                          - - AATAAAATTC GCGTTCTTTT TACG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 26:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: primer pcrfl - #o2                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                          - - GAGCTCAAGC TTCGTAAAAA GAACGCGAAT T        - #                  - #              31                                                                    __________________________________________________________________________

We claim:
 1. A method for immobilizing an enzyme comprisingrecombinanltly producing an enzyme or a functional fragment thereoflinked to the exterior of a host cell, said method comprising localizingthe enzyme or functional fragment thereof at the exterior of the cellwall of a fungus by linking the enzyme or the functional part thereof tothe anchoring part of a cell wall anchoring protein, which anchoringpart is derivable from the C-terrmal part of said anchoring protein. 2.A method according to claim 1 in which said fungus is a yeast.
 3. Themethod of claim 1, in which said fungus is selected from the groupconsisting of yeasts belonging to the genera Candida, Debaryomyces,Hansenula, Kluyveromyces, Pichia and Saccharomyces, and molds belongingto the genera Aspergillus, Penicillium and Rhizopus.
 4. A funguscontaining an expressible first polynucleotide comprising a structuralgene encoding a protein providing catalytic activity and at least a partof a gene encoding at least a C-terminal portion of an anchoring proteincapable of anchoring in the cell wall of said fungus, said part encodingar least the anchoring part of said anchoring protein, said firstpolynucleotide being present either in a vector or in the chromosome ofsaid fungus.
 5. The fungus of claim 4, further comprising a sequenceencoding a signal peptide, said sequence being operably linked to saidfirst polynucleotide such that the translation product of said firstpolynucleotide is secreted to the cell wall of said fungus.
 6. Thefungus of claim 5, wherein the signal peptide is derived from a proteinselected from the group consisting of glycosyl-phosphatidyl-inositol(GPI) anchoring protein, α-factor, α-agglutinin, a-agglutinin, invertaseor inulinase of yeasts, α-amylase of Bacillus, and proteinases of lacticacid bacteria.
 7. The fungus of claim 4, wherein the protein capable ofanchoring in the cell wall of said fungus is selected from the groupconsisting of α-agglutinin, a-agglutinin, flocculation protein, andMajor Cell Wall Protein of a fungus.
 8. The fungus of claim 4, whereinthe protein providing catalytic activity is selected from the groupconsisting of a hydrolytic enzyme and an oxido-reductase.
 9. The fungusof claim 8 wherein said hydrolytic enzyme is a lipase.
 10. The fungus ofclaim 8 wherein said protein providing catalytic activity is an oxidase.11. The fungus of claim 4, wherein the protein providing catalyticactivity exhibits said catalytic activity when present in a multimericform, said fungus further comprising a second polynucleotide comprisinga structural gene encoding said protein providing catalytic activityoperably linked to a sequence encoding a signal peptide ensuringsecretion of the expression product of said second polynucleotide whichis operably linked to a regulatable promoter.
 12. The fungus of claim11, wherein said second polynucleotide is present either in a separatevector than the first polynucleotide or is present in the chromosome ofsaid fungus.
 13. The fungus of claim 4, having at least one of saidpolynucleotides integrated in its chromosome.
 14. The fungus of claim 4,having said protein providing catalytic activity immobilized at theexterior of its cell wall.
 15. The fungus of claim 4, which is a yeast.16. A process for carring out an enzymatic process by using animmobilized catalytically active protein, wherein a substrate for saidcatalytically active protein is contacted with the fungus of claim 4.17. A process according to claim 16 in which the fungus is a yeast.